Supplementary Figures Supplementary Tables 1 and 2. Supplementary References

Transcription

1 The Nuclear Import of a Non-Canonical Frizzled2 Signal by Importins-β11 and α2 Promotes Postsynaptic Development Timothy J. Mosca and Thomas L. Schwarz * The F.M. Kirby Neurobiology Center, Children s Hospital Boston, 300 Longwood Avenue, Boston, Massachusetts, USA and Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, Massachusetts, USA Present Address: Dept. of Biology, Stanford University, 385 Serra Mall, Stanford, CA, * Correspondence should be addressed to TLS (thomas.schwarz@childrens.harvard.edu) Supplementary Figures 1 13 Supplementary Tables 1 and 2 Supplementary References

2 a Fz2-C c Fz2-C d Fz2-C HRP WT b Fz2-C dfz2 C1 / Df(3L)ED4782 WT WT e FLAG g FLAG f Ab Control FLAG Muscle Fz2-FLAG Muscle Fz2-FLAG Supplementary Figure 1. Fz2-C is Imported into Larval Muscle Nuclei. (a) Representative confocal image of a wild-type (y,w; FRT42D; ; ) third instar larval muscle nucleus stained with an anti-fz2-c antibody. Punctate, immunoreactive structures are visible within the nucleus (dashed circle). (b) A dfz2 mutant nucleus (y,w; ; dfz2 C1 / Df(3L)ED4782; ) stained as in (a). The absence of staining indicates that the antibody is specifi c for Fz2. Scale bar = 10 μm. (c,d) A wild-type third instar NMJ at muscles 6 and 7 stained with antibodies against Fz2-C (magenta) and HRP (green). Synaptic staining of the Fz2 receptor was observed, con-

3 fi rming the results of Mathew et al., (2005). (e,f) Muscle nuclei stained with anti-flag antibodies lack FLAG immunoreactivity (green) in wild-type (e), when no FLAG transgene is expressed but contain immunoreactive puncta (f) when expressing a C-terminally FLAG-tagged Fz2 construct in the body-wall muscles (y,w; UAS-Fz2-FLAG / ; 24B-GAL4 / ; ) (g) FLAG immunoreactivity is also evident postsynaptically at the NMJ. Thus FLAG-tagged Fz2 localizes similarly to the endogenous DFz2 and offers independent confi rmation of the model of Mathew and colleagues 1. In all cases, scale bar = 10 μm.

4 a Fz2-C b Fz2-C Neuron Wg β11 -/- Neuron Wg c Fz2-C d Fz2-C β11 -/- β11 -/- Muscle β11 e Fz2-C f Fz2-C α2 -/- α2 -/- Muscle α2 Supplementary Figure 2. Nuclear Localization of Fz2-C can be Rescued with Muscle Expression of the Importins, but not by Wingless Overexpression. Anti-Fz2-C staining of third-instar muscle nuclei in the following genotypes: (a) Neuron Wg = y,w; ; elav-gal4 / UAS-Wingless;, (b) β11 -/- Neuron Wg = y,w; imp-β11 70 / Df; elav-gal4 / UAS- Wingless;, (c) β11 -/- = y,w; imp-β11 70 / Df; ;, (d) β11 -/- Muscle β11 = y,w; imp-β11 70 / Df; 24B-GAL4 / UAS-Importin-β11-eGFP;, (e) α2 -/- = y,w; imp-α2 D14 ; ; and (f) α2 -/- Muscle α2 = y,w; imp-α2 D14 ; 24B-GAL4 / UAS-Importin-α2;. Wingless overexpression in the motoneuron increased nuclear Fz2-C puncta (a) consistent with increased activation of muscle Fz2 receptors. This activation was insuffi cient to overcome the defi cit in nuclear Fz2-C in the imp-β11 mutant (b). The absence of nuclear puncta in imp-β11 (c) or imp-α2 (e) mutants, is rescued by their transgenic restoration (d,f) to the muscles. Scale bar = 10 μm.

5 a Wild-Type b imp-β11 70 / Df c dl 1 Imp-β1 (Ketel) Importin-α2 Muscle nls-gfp nona Dorsal d f h j e g i k Supplementary Figure 3. Import of Many Nuclear Cargoes is Not Impaired in importinβ11 Mutants. (a-c) Representative single sections through the center of third instar larval muscle nuclei and stained with an antibody to Dorsal, an NF-κB transcription factor and synapse-to-nucleus signalling molecule. Nuclear staining is observed in wild-type (a: y,w; FRT42D; ; ) and in imp-β11

6 mutants (b: y,w; imp-β11 70 / Df; ; ), but absent in dorsal mutants (c: y,w; dl 1 ; ; ), indicating that the nuclear staining is specifi c for Dorsal and independent of Importin-β11. (d,e) Wild-type and imp-β11 mutant muscle nuclei are both immunoreactive for an antibody to nona, an RNAbinding protein expressed in all Drosophila nuclei. (f,g) A GFP-tagged transgene with a nuclear localization signal expressed in the muscle of wild-type (y,w; FRT42D; 24B-GAL4 / UAS-NLS- GFP; ) and imp-β11 mutants (y,w; imp-β11 70 / Df; 24B-GAL4 / UAS-NLS-GFP; ). In both cases, the NLS-GFP construct is imported into muscle nuclei. (h,i) Wild-type and imp-β11 mutant muscle nuclei stained with an antibody to Importin-α2. Importin-α2 nuclear localization does not require Importin-β11. (j,k) Wild-type and imp-β11 mutant muscle nuclei stained with an antibody to Ketel, the Drosophila homologue of Importin-β1. Import of Ketel also does not require Importin-β11. Thus the lack of nuclear Fz2-C in imp-β11 mutant nuclei does not refl ect a general block of nuclear import. Scale bar = 10 μm.

7 a Futsch / MAP1b (22C10) HRP g Wild-Type b imp-α2 D14 imp-β11 70 / Df c d e f h Supplementary Figure 4. Futsch Staining is Normal in Importin Mutants. (a-f) Representative confocal images of third-instar larval NMJs stained with antibodies to Futsch (green) and HRP (magenta) to assess organization of the presynaptic microtubule cytoskeleton. As in wild-type larvae (a,b: y,w; FRT42D; ; ), similar patterns of synaptic staining and microtubule loops (insets) are observed in imp-β11 mutants (c,d: y,w; imp-β11 70 / Df; ; ) and impα2 mutants (e,f: y,w; imp-α2 D14 ; ; ). Scale bar = 10 μm for full images and 1.5 μm for insets. (g) Quantifi cation of the % of boutons with Futsch loops in the above genotypes. There is no signifi cant difference among the genotypes. Specifi c values were as follows: WT = 20% ± 1.0%, n = 8 animals, 15 NMJs; imp-β11 70 / Df = 20% ± 0.91%, n = 8 animals, 15 NMJs; imp-α2 D14 = 18% ± 0.52%, n = 8 animals, 16 NMJs, p > 0.1 for all comparisons. (h) Quantifi cation of the % of boutons with unbundled Futsch in the above genotypes found no signifi cant differences across the genotypes: WT = 9.7% ± 1.4%, n = 8 animals, 15 NMJs; imp-β11 70 / Df = 8.7% ± 0.98%, n = 8 animals, 15 NMJs; imp-α2 D14 = 9.0% ± 0.47%, n = 8 animals, 16 NMJs, p > 0.1 for all cases.

8 a HRP Discs Large (Dlg) b c j d e f wingless TS Wild-Type dfz2 C1 / Df(3L)ED4782 g h i Supplementary Figure 5. Loss of Wingless or Fz2 Increases Ghost Boutons at the NMJ. (a-c) Wild-type (y,w; FRT42D; ; ) third instar larval NMJs stained with anti-hrp (magenta) and anti-dlg (green). Each wild-type presynaptic bouton faces a postsynaptic accumulation of DLG. (d-i) wingless mutant (y,w; wg TS ; ; ) and dfz2 mutant (y,w; ; dfz2 C1 / Df(3L)ED4782; ) NMJs stained as above. Though many boutons appear normal, there is an increased frequency of ghost boutons with HRP immunoreactivity but no opposite postsynaptic DLG (arrows). Scale bar = 5 μm. (j) Quantifi cation of ghost boutons at larval NMJs. Mutant conditions are indicated by the color and shading of the bar and any expressed transgenes are indicated below the X-axis. Loss of either Wingless or Fz2 results in a 3-4 fold increase in ghost boutons; the dfz2 mutant phenotype can be rescued by muscle restoration of NLS-Fz2-C expression in the mutant background. A viable wingless hypomorph results in an intermediate phenotype. Genotypes and specifi c values are as follows: Wild-Type or / (y,w; FRT42D; ; ) = 1.0 ± 0.18 ghost boutons / NMJ, n = 10 animals, 59 NMJs; Neuron Wg (y,w; FRT42D; elav-gal4 / UAS-Wingless; ) = 0.54 ± 0.13, n = 12 animals, 71 NMJs; eag, Sh -/- (eag 1 Sh KS133 ; ; ; ) = 0.59 ± 0.10, n = 11 animals, 63 NMJs; wg hypo (y,w; wg 1 ; ; ) = 2.1 ± 0.27 ghost boutons / NMJ, n = 8 animals, 46 NMJs; wg null (y,w;

9 wg TS ; ; ) = 3.9 ± 0.41 ghost boutons / NMJ, n = 10 animals, 56 NMJs; dfz2 -/- (y,w; ; dfz2 C1 / Df(3L)ED4782; ) = 3.1 ± 0.32 ghost boutons / NMJ, n = 14 animals, 76 NMJs; dfz2 -/- Muscle NLS-Fz2-C (w, UAS-myc-NLS-Fz2-C; BG487-GAL4 / ; dfz2 C1 / Df(3L)ED4782; ) = 0.71 ± 0.24, n = 3 animals, 17 NMJs, p vs. / > 0.2, vs. dfz2 -/- < ). Furthermore, manipulations that result in increased nuclear Fz2-C puncta (eag, Sh mutants and neuronally overexpressed Wingless) result in a signifi cant decrease of ghost boutons compared to wild-type. Scale bar = 5 μm. * p < 0.05 and *** p <

10 a b c Supplementary Figure 6. Loss of Nuclear Fz2-C Alters SSR Area Without Altering Presynaptic Bouton Area. (a) Quantification of presynaptic bouton area from electron micrographs in Wild-Type (y,w; FRT42D; ; ), dfz2 mutant (dfz2 -/- = y,w; ; dfz2 C1 / Df(3L)ED4782; ), imp-β11 mutant (β11 -/- = y,w; imp-β11 70 / Df; ; ), and imp-β11 mutant larvae expressing NLS-Fz2-C in the musculature (β11 -/- Muscle NLS-Fz2-C = w, UAS-myc-NLS-Fz2-C; imp-β11 70 / Df; 24B-GAL4 / ; ). Bouton area is not signifi cantly different (p > 0.1) from wild-type in any of the experimental genotypes. Specifi c values are as follows: Wild-Type = 2.45 ± μm 2, n = 5 animals, 52 boutons; β11 -/- = 1.96 ± μm 2, n = 5 animals, 17 boutons; dfz2 -/- = 2.10 ± μm 2, n = 3 animals, 26 boutons; β11 -/- Muscle NLS-Fz2-C = 2.98 ± μm 2, n = 5 animals, 104 boutons. (b) Quantifi - cation of area of the SSR in the above genotypes. The area of the SSR is signifi cantly decreased by nearly 60% in both imp-β11 and dfz2 mutant larvae (p < for both cases). This reduction is completely rescued by restoring nuclear Fz2-C to the imp-β11 mutant (p > 0.4). Specific values are as follows: Wild-Type = 7.90 ± μm 2, n = 5 animals, 52 boutons; β11 -/- = 3.50 ± μm 2, n = 5 animals, 17 boutons; dfz2 -/- = 3.76 ± μm 2, n = 3 animals, 26 boutons; β11 -/- Muscle NLS-Fz2-C = 7.72 ± μm 2, n = 5 animals, 104 boutons. (c) Quantifi cation of the ratio of the area of the SSR to the area of the bouton in the above genotypes. The ratio of SSR area to bouton area is signifi cantly decreased by nearly 40% in both mutant genotypes (p < 0.001) and restored to wild-type levels by restoring nuclear Fz2-C (p > 0.6). Specifi c values are as follows: Wild-Type = 3.85 ± 0.251, n = 5 animals, 52 boutons; β11 -/- = 2.21 ± 0.282, n = 5 animals, 17 boutons; dfz2 -/- = 2.33 ± 0.351, n = 3 animals, 26 boutons; β11 -/- Muscle nls-fz2-c = 3.68 ± 0.327, n = 5 animals, 104 boutons. **, p < 0.001, *** p <

11 Mosca and Schwarz, 2010 a c imp-β1170 / Df b β11 -/- Muscle NLS-Fz2-C d dfz2c1 / Df(3L)ED4782 β11 -/- Muscle NLS-Fz2-C Supplementary Figure 7. EM of Subsynaptic Reticulum in Additional Mutant and Rescue Genotypes. (a-d) TEM of type Ib boutons of NMJs at muscles 6 and 7 in segment A2 in imp-β11 mutants (a: y,w; imp-β1170 / Df; ; ), dfz2 mutants (b: y,w; ; dfz2c1 / Df(3L)ED4782; ) and imp-β11 mutants expressing myc-nls-fz2-c in the muscle (c,d: β11 -/- Muscle NLS-Fz2-C = w, UAS-myc-NLSFz2-C; imp-β1170 / Df; 24B-GAL4 / ; ). In these images, the SSR is false colored in red. The examples in (a) and (b) represent the ultrastructural correlates of what may have been scored as ghost boutons at the confocal level in the imp-β11 and dfz2 mutants. In the case of (b), there is no SSR, suggesting that this is the true ultrastructural correlate of a ghost bouton. Both (c) and (d) are representative examples of the SSR rescue observed when expressing myc-nls-fz2-c in imp-β11 mutant muscles. In these cases, robust SSR is restored to each synaptic bouton. In all images, scale bar = 500 nm.

12 GluRIIA GluRIIB GluRIIC A B C Wild-Type D E F imp-β11 70 / Df G H I Supplementary Figure 8. Intensity of Anti-Glutamate Receptor Immunostaining is Unaffected by Loss of Importin-β11. (a-f) Representative confocal images of third-instar larval NMJs stained with antibodies to glutamate receptor subunits GluRIIA (a, d), GluRIIB (b, e) or GluRIIC (c, f). Both wild-type (a-c: y,w;

13 FRT42D; ; ) and imp-β11 mutant (d-f: y,w; imp-β11 70 / Df; ; ) larvae have each sub-type of glutamate receptor localized at synapses. Scale bar = 10 μm. (g-i) Quantifi cation of the fl uorescent intensity of immunoreactivity for GluRIIA (g), GluRIIB (h) and GluRIIC (i) in wild-type and imp-β11 mutant larvae. No statistically signifi cant differences were measured. In all cases, p > 0.7 and n = 6 animals, 12 NMJs.

14 dpak HRP Wild-Type imp-β11 70 / Df a b c d e dpix HRP f g h i j pdlg S797 HRP PAR-1 HRP Synd HRP k l m p q r s u v w x n o t y WASp HRP z a b c d Supplementary Figure 9. Candidate Subsynaptic Reticulum (SSR)-Related Molecules are Unchanged in importin-β11 Mutants. Representative confocal stack images of NMJs at muscle 4 stained with antibodies to HRP (magenta) and dpak (green: a-d), dpix (green: f i), PAR-1 (green: k-n), pdlgs797 (green: p-s),

15 Syndapin (green: u-x) or WASp (green: z-c ). These proteins are known to affect the thickness of the SSR either through loss of function or overexpression effects. For both wild-type (y,w; FRT42D; ; ) and imp-β11 (y,w; imp-β11 70 / Df; ; ) mutant larvae, each protein is properly enriched at the NMJs. Scale bar = 10 μm. (e,j,o,t,y,d ) and quantitative comparison of their fl uorescent intensity in wild-type and imp-β11 larvae reveals no signifi cant differences. In all cases, p > 0.1 and n 4 animals, 12 NMJs.

16 α-spectrin HRP Wild-Type a type Is b type Is e f type Ib type Ib imp-β11 70 / Df c d g type Ib type Ib type Is type Is h i j k l Supplementary Figure 10. Thickness and Fluorescence Intensity of α-spectrin Immunoreactivity is Reduced Surrounding Boutons of importin-β11 Mutants. (a d) Representative confocal stack images of NMJs at muscle 4 stained with antibodies to HRP (magenta) and α-spectrin (green) in wild-type (y,w; FRT42D; ; ) and imp-β11 (y,w; impβ11 70 / Df; ; ) mutant larvae. The intensity of α-spectrin staining is reduced in the imp-β11 mutant while HRP intensity appears unchanged. (e h) Representative single slice high magnifi cation images of boutons at muscle 4 stained as in (a d). In imp-β11 larvae (g,h), the width of the α-spectrin staining surrounding type Ib boutons is reduced compared to wild-type controls (e,f). Scale bars = 10 μm. (i) Quantifi cation of the fl uorescence intensity of α-spectrin in wild-type and imp-β11 mutants reveals a 23% reduction in imp-β11 mutants. Here, p < and n = 6 animals, 16 NMJs for each genotype. (j l) Quantifi cation of the widths of α-spectrin (j), HRP (k)

17 and the ratio of α-spectrin width to HRP width (l) surrounding type Ib boutons in both wild-type and imp-β11 mutants. α-spectrin width was reduced by 42% in the imp-β11 mutant while HRP width is unchanged across genotypes. Consistent with this, the ratio of α-spectrin width to HRP width is also decreased by 40%. Specifi c values are as follows: WT α-spectrin width = 1.80 ± μm, HRP width = 3.44 ± μm, α-spectrin Width / HRP width = ± ; impβ11 70 / Df α-spectrin width = 1.03 ± μm, HRP width = 3.27 ± μm, α-spectrin width / HRP width = ± For α-spectrin width and α-spectrin width / HRP width, p < For HRP width, p > 0.4. For all cases, n = 4 animals, 8 NMJs, 32 boutons.

18 a Fz2-8xMyc dgrip-v5 Input IP (anti-myc) V5 Full-Length Fz2-8xMyc Myc IgG Heavy Chain Fz2-8xMyc C-Terminus α-tubulin b Fz2-8xMyc Imp-β11-eGFP GFP Input IP (anti-myc) Full-Length Fz2-8xMyc Myc IgG Heavy Chain Fz2-8xMyc C-Terminus α-tubulin Supplementary Figure 11. Fz2 and dgrip, but not Fz2 and Importin-β11, Co-Immunoprecipitate in S2 Cells. (a) Immunoblots of S2 cells transfected with Fz2-8x-Myc and dgrip-v5. The proteins were immunoprecipitated with anti-myc antibodies and detected with anti-v5 or anti-myc antibodies. The dgrip scaffolding protein is coimmunoprecipitated with the Fz2 receptor when the two are coex-

19 pressed but not when dgrip is expressed alone. Anti-Myc antibodies detect both full length and C-terminal truncation species of Fz2 (arrows). A band corresponding to IgG heavy chain was also detected in all IP lanes. Alpha-tubulin was used as a loading control. (b) Immunoblots of S2 cells transfected with Importin-β11-eGFP and Fz2-8x-Myc. Proteins were again immunoprecipitated with anti-myc antibodies and detected with anti-gfp or anti-myc antibodies. Under these conditions, Importin-β11-eGFP is not observed to co-immunoprecipitate with the Fz2 receptor. Again, the anti-myc antibodies detected multiple species of Fz2 as labeled (arrows) and alphatubulin was used as a loading control. All blots were cropped; the full-length blots are presented in Supplementary Figure 13.

20 a b Nerve Nerve Wild-Type X Muscle? nucleus Muscle SSR Wg Imp-α2 Imp-β11 Fz2 Fz2-C nls-fz2-c? nucleus Importin Mutant X c Nerve Muscle? nucleus nls-fz2-c Rescue X Supplementary Figure 12. Nuclear Import of Fz2-C by Importin β11 and Importin α2 Enables Proper Development of the Postsynaptic SSR A proposed model for the activity-dependent regulation of SSR growth by a synapse to nuclear signal: (a) Wg is released from presynaptic terminals and activates Fz2 receptors at the synapse. Importins-β11 and -α2 interact with the Fz2 receptor at the synapse and, after receptor cleavage, accompany the C-terminus peptide to the nuclear pore and facilitate its nuclear entry. Once inside, the C-terminus activates gene expression that promotes development of the postsynapse, including the subsynaptic reticulum and its associated protein Discs Large. (b) When the import of Fz2-C is blocked in importin-β11 and importin-α2 mutants, gene expression is not activated and less SSR forms, and some boutons lack Discs Large. Neither endocytosis nor presence of the Fz2-C peptide in cytoplasmic puncta are impaired in importin mutants, however. (c) When normal Fz2-C import is thus blocked, postsynaptic development can be restored by expression of the NLS-tagged Fz2-C, whose nuclear import is independent of the importins.

21 4j) Input: anti-gfp 4j) IP: anti-gfp Imp-β11-eGFP Fz2-FLAG Input Imp-β11-eGFP Fz2-FLAG IP (anti-flag) 250 kda 150 kda 100 kda 75 kda 50 kda 250 kda 150 kda 100 kda 4j) Input: anti-flag 4j) IP: anti-flag 4j) Input: anti-α-tubulin Imp-β11-eGFP Fz2-FLAG Input Imp-β11-eGFP Fz2-FLAG IP (anti-flag) Imp-β11-eGFP Fz2-FLAG Input 250 kda 150 kda 100 kda 75 kda 50 kda 37 kda 25 kda 20 kda 250 kda 150 kda 100 kda 75 kda 50 kda 37 kda 25 kda 20 kda 50 kda 37 kda S11a) Input: anti-α-tubulin Fz2-8xMyc dgrip-v5 Input 50 kda 37 kda S11a) Input: anti-myc Fz2-8xMyc dgrip-v5 250 kda 150 kda 100 kda 75 kda 50 kda 37 kda Input S11a) IP: anti-myc Fz2-8xMyc dgrip-v5 250 kda 150 kda 100 kda 75 kda 50 kda 37 kda IP (anti-myc) S11a) Input: anti-v5 Fz2-8xMyc dgrip-v5 250 kda 150 kda 100 kda 75 kda 50 kda 37 kda 25 kda Input Supplementary Figure 13a. Full Blots from Figure 4 and Supplementary Figure 11

22 S11a) IP: anti-v5 S11b) Input: anti-myc S11b) IP: anti-myc IP (anti-myc) Input IP (anti-myc) Fz2-8xMyc dgrip-v5 Fz2-8xMyc Imp-β11-eGFP Fz2-8xMyc Imp-β11-eGFP 150 kda 100 kda 75 kda 50 kda 250 kda 150 kda 100 kda 75 kda 250 kda 150 kda 100 kda 75 kda 37 kda 50 kda 50 kda 25 kda 37 kda 37 kda S11b) Input: anti-gfp S11b) IP: anti-gfp S11b) Input: anti-α-tubulin Input IP (anti-myc) Input Fz2-8xMyc Imp-β11-eGFP 150 kda 100 kda 75 kda Fz2-8xMyc Imp-β11-eGFP 250 kda 150 kda 100 kda Fz2-8xMyc Imp-β11-eGFP 50 kda 37 kda 75 kda Supplementary Figure 13b. Full Blots from Figure 4 and Supplementary Figure 11

23 Supplementary Table 1. Alleles and Transgenic Fly Strains Used in this Study Allele Chromosome Supplemental Reference Df(2R) m22 II [1, 2] Df(3L)ED4782 III [3] Df(3L) 1S1 III [4] dfz2 C1 II [5] dl 1 II [6] eag 1 Sh KS133 X [7] FRT42D II [8] imp- 2 D14 II [9] imp- 2 D3 II [9] imp II [2] wg 1 II [10] wg IL114 (wg TS ) II [11] wit A12 III [12, 13] wit B11 III [12, 13] GAL4 Driver Chromosome Supplemental Reference 24B-GAL4 III [14] BG487-GAL4 II [15] Elav-GAL4 III [16] UAS Construct Chromosome Supplemental Reference UAS-DFz2-FLAG II [17] UAS-Importin- 2 III [18] UAS-Importin- 11-eGFP III [2] UAS-myc-NLS-DFz2-C X [3] UAS-NLS-GFP III [19] UAS-Wingless III [20]

24 Supplementary Table 2. Antibodies Used for Immunocytochemistry in This Study Antigen Animal Dilution Supplemental Reference -spectrin (3A9) Mouse 1:50 [21, 22] DFz2-C Rabbit 1:200 [3, 23] DFz2-N Rabbit 1:100 [3, 23] dgrip Rat 1:200 [24] DLG (4F3) Mouse 1:500 [25] Dorsal Rabbit 1:1000 [26] dpak Rabbit 1:500 [27] dpix Mouse 1:25 [25] FLAG Rabbit 1:500 Commercial, Sigma-Aldrich Futsch Mouse 1:50 [28-30] GluRIIA Mouse 1:100 [31] GluRIIB Rabbit 1:2500 [32] GluRIIC Rabbit 1:2000 [32] Importin- 1 Rabbit 1:100 [33] Importin- 2 Rabbit 1:100 [33] Importin- 3 Rabbit 1:100 [34] Importin- 11 Rabbit 1:750 [2] Ketel Rabbit 1:200 [35] Lamin C Mouse 1:200 [36] Myc Mouse 1:300 Commercial, Santa Cruz Nanos Rat 1:300 [37] nona Mouse 1:100 [38] PAR-1 Rabbit 1:100 [39, 40] pdlg S797 Rabbit 1:200 [40] Syndapin Guinea Pig 1:500 [41] WASp Guinea Pig 1:1000 [41] Wingless Rabbit 1:200 [23, 42]

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