Fig 1: Morphological changes in vesicular trafficking compartments in Klhl40a deficient myofibers.Sar1a is increased and co-localized with PDI in Klhl40a deficiency. The number of PDI-positive foci is also increased in the absence of Klhl40a. The fraction of Sec23 and Tango1 positive foci is decreased in klhl40a mutant myofibers. Disruption of the Golgi architecture was observed in a fraction of Klhl40a deficient myofibers. Data are mean ± S.E.M (unpaired t-test, parametric) for each quantification.
Fig 2: Klhl40a loss results in perturbation of the ER-Golgi vesicle trafficking through secretion-associated Ras GTPase (Sar1a).(A) ER-Golgi vesicle trafficking proteins exhibit altered levels in klhl40a mutant muscle compared to control (+/+) in proteome analysis; ns indicates no significant difference (B) Western blot showing ER-exit site protein Sar1a is upregulated in klhl40a mutant muscle, and downstream COPII and Golgi proteins are downregulated in mutant muscle (3mo) (C) Quantification of the protein by Western blot in klhl40a and control zebrafish. N=3 in each group. Data are mean ± S.E.M; with one-way ANOVA and Tukey’s HSD test (****p<0.001). (D) Transmission electron microscopy (TEM) of zebrafish larva (4 dpf) with SAR1A mRNA overexpression demonstrating abnormal membrane structures in the SR-ER region. (E) Immunofluorescence of control and SAR1A overexpressing myofibers (5 dpf); n=25–30 myofibers in each group. Figure 5—source data 1.Full unedited Sar1a immunoblot. Figure 5—source data 2.Full unedited Sec24d immunoblot. Figure 5—source data 3.Full unedited Golga2 immunoblot. Figure 5—source data 4.Full unedited a-tubulin immunoblot. Figure 5—source data 5.Annotated immunoblots. Figure 5—source data 6.Proteomic data for autophagy markers.
Fig 3: SAR1A is a direct ubiquitylation target of the KLHL40-CUL3 complex and is differently ubiquitylated by a disease-causing mutation in KLHL40.(A) Coimmunoprecipitation in C2C12 cells showing KLHL40 directly interacts with SAR1A. (B) Co-overexpression of decreasing KLHL40-FLAG and constant SAR1A-V5 in C2C12 myoblasts demonstrates that KLHL40 is a regulator of Sar1A protein. (C) Co-overexpression of decreasing amounts of KLHL40-FLAG and constant amount of SAR1A-V5 in C2C12 myoblasts in the presence of UPS inhibitor MG132 increases the SAR1A protein levels in comparison to MG132- condition. (D) Alignment of the amino acid sequence of the SAR1A ubiquitylation site demonstrates high conservation in vertebrates (K182 in all species, marked by the asterisk). (E) Localization of different disease-causing variants in KLHL40 in the protein domains. (F) In vitro ubiquitylation of human SAR1A by CUL3 protein complex in the presence of wild-type and disease-causing KLHL40 proteins. (G) Quantifying the relative human SAR1A ubiquitylation by wild-type and disease-causing KLHL40-CUL3 complex. (H) Ubiquitylation of overexpressed SAR1A in the presence of KLHL40 in C2C12 myoblasts. Data are mean ± S.E.M; with one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test and Brown-Forsythe test (****p<0.001; n.s. non significant) n=3. Figure 7—source data 1.Full unedited 7A immunoblot with FLAG antibody. Figure 7—source data 2.Full unedited 7A immunoblot with V5 antibody. Figure 7—source data 3.Full unedited 7B immunoblot with FLAG antibody. Figure 7—source data 4.Full unedited 7B immunoblot with V5 antibody. Figure 7—source data 5.Full unedited 7B immunoblot with GAPDH antibody. Figure 7—source data 6.Full unedited 7C immunoblot with FLAG antibody. Figure 7—source data 7.Full unedited 7C immunoblot with V5 antibody. Figure 7—source data 8.Full unedited 7C immunoblot with GAPDH antibody. Figure 7—source data 9.Full unedited 7F immunoblot with SAR1A antibody. Figure 7—source data 10.Full unedited 7F protein gel. Figure 7—source data 11.Full unedited 7H immunoblot with FLAG antibody. Figure 7—source data 12.Full unedited 7H immunoblot with SAR1A antibody. Figure 7—source data 13.Annotated immunoblots and gel.
Supplier Page from Abcam for Anti-SAR1 antibody