Journal: Genome Medicine

Article Title: Linkage, whole genome sequence, and biological data implicate variants in RAB10 in Alzheimer’s disease resilience

doi: 10.1186/s13073-017-0486-1

Figure Lengend Snippet: Replication test results in the CCS for selected SNPs

Article Snippet: Taqman (Thermo Fisher Scientific) real time PCR assays were used to measure the expression of RAB10 (Mm00489481_m1), SAR1A (Mm01150424_m1), and the housekeeping gene GAPDH (Hs02758991_g1).

Techniques:

Journal: Genome Medicine

Article Title: Linkage, whole genome sequence, and biological data implicate variants in RAB10 in Alzheimer’s disease resilience

doi: 10.1186/s13073-017-0486-1

Figure Lengend Snippet: Mayo Clinic Brain RNAseq data for SAR1A and RAB10 genes in AD vs. control brains

Article Snippet: Taqman (Thermo Fisher Scientific) real time PCR assays were used to measure the expression of RAB10 (Mm00489481_m1), SAR1A (Mm01150424_m1), and the housekeeping gene GAPDH (Hs02758991_g1).

Techniques: Control, Comparison

Journal: Genome Medicine

Article Title: Linkage, whole genome sequence, and biological data implicate variants in RAB10 in Alzheimer’s disease resilience

doi: 10.1186/s13073-017-0486-1

Figure Lengend Snippet: Sar1a subtly affects APP processing in vitro. a Full-length APP levels are not altered by Sar1a expression. Immunoblots of N2A695 cells transiently transfected with vectors expressing GFP, Sar1a, scrambled shRNA, or shRNA specific to Sar1a. Open arrowhead , APP ; closed arrowhead , CTF-β and CTF-ɑ. b Sar1a expression does not significantly alter extracellular amyloid-beta levels. Conditioned media from N2A695 cells overexpressing or silencing Sar1a were analyzed by ELISA and resulting values were expressed relative to control. c Sar1a expression does not significantly alter sAPP levels. Quantification of immunoblots of sAPPalpha, sAPPbeta, and sAPPtotal. d Sar1a overexpression alters CTF-β and CTF-ɑ. Quantification of immunoblots of full-length APP , CTF-β, and CTF-ɑ. Graphs represent mean ± SEM from at least three independent experiments. * p value < 0.05

Article Snippet: Taqman (Thermo Fisher Scientific) real time PCR assays were used to measure the expression of RAB10 (Mm00489481_m1), SAR1A (Mm01150424_m1), and the housekeeping gene GAPDH (Hs02758991_g1).

Techniques: In Vitro, Expressing, Western Blot, Transfection, shRNA, Enzyme-linked Immunosorbent Assay, Control, Over Expression

Journal: Genome Medicine

Article Title: Linkage, whole genome sequence, and biological data implicate variants in RAB10 in Alzheimer’s disease resilience

doi: 10.1186/s13073-017-0486-1

Figure Lengend Snippet: Variants in the linkage region after filtering

Article Snippet: Taqman (Thermo Fisher Scientific) real time PCR assays were used to measure the expression of RAB10 (Mm00489481_m1), SAR1A (Mm01150424_m1), and the housekeeping gene GAPDH (Hs02758991_g1).

Techniques: Variant Assay

Journal: Scientific Reports

Article Title: The role of Rab6a and phosphorylation of non-muscle myosin IIA tailpiece in alcohol-induced Golgi disorganization

doi: 10.1038/srep31962

Figure Lengend Snippet: ( a ) Immunostaining of giantin in VA-13 cells (HepG2 cells expressing ADH). First row: no treatment (Ctrl), EtOH treatment (35 mM for 72 h) and EtOH treatment followed by 25 μM blebbistatin at 48 h; second row: control siRNA, and 35 mM EtOH for 72 h plus control or MYH9 siRNAs; third row: MYH9 siRNAs, SAR1A siRNAs or SAR1A + MYH9 siRNAs. The right panel shows high magnifications of the highlighted area (white boxes). Nuclei were counterstained with DAPI (blue). ( b ) NMIIA Western blot of the lysates of VA-13 cells treated with 35 mM EtOH for 72 h; β-actin was a loading control. ( c ) Quantitative real-time PCR analysis of the mRNA of NMIIA in control VA-13 cells and treated with 35 mM EtOH for 72 h. Data were presented as a mean from the three independent experiments and expressed as the fold relative to that (100%, 1 fold) of GAPDH. ( d ) NMIIA Western blot of the lysates of VA-13 cells treated with scramble or MYH9 siRNAs; β-actin was a loading control. ( e ) NMIIA and Sar1a Western blot of the lysates of VA-13 cells treated with scramble or S AR1A plus NMIIA siRNAs; β-actin was a loading control. ( f ) Quantification of the fragmented Golgi in cells treated as described in the ( a ) n = 60 cells from three independent experiments.

Article Snippet: MYH9 (myosin, heavy polypeptide 9, non-muscle, NMIIA), SAR1A and scrambled on-targetplus smartpool siRNAs were purchased from Santa Cruz Biotechnology.

Techniques: Immunostaining, Expressing, Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: The role of Rab6a and phosphorylation of non-muscle myosin IIA tailpiece in alcohol-induced Golgi disorganization

doi: 10.1038/srep31962

Figure Lengend Snippet: ( a,b ) Immunostaining of Rab6a (green) and giantin (red) in ( a ) VA-13 cells: control, EtOH- or SAR1A siRNA-treated; ( b ) immunostaining of giantin (green) and Rab6a (red) in hepatocytes isolated from control and EtOH-fed rats. The right panels show green and red channels corresponding to Golgi and cytoplasmic regions (white boxes). Nuclei were counterstained with DAPI (blue). All confocal images were acquired with same imaging parameters; bars, 10 μm. ( c ) Quantification summarizing the Rab6a-specific fluorescence signal colocalized with giantin in cells presented in ( a , b ). The Pearson’s coefficient is presented as a mean ± SD from three independent experiments; *p < 0.001. ( d ) Immunostaining of giantin in VA-13 cells transiently transfected with empty PCMV vector (Ctrl), treated with 35 mM EtOH for 72 h and simultaneously transiently transfected with empty PCMV vector (EtOH), or transfected with Rab6(T27N) or NMIIAΔtailpiece plasmids. ( e ) Quantification of the fragmented Golgi in cells treated as described in the ( d ) n = 60 cells from three independent experiments. ( f ) NMIIA-P-S1943 Western blot of the Golgi fraction isolated from hepatocytes of control and EtOH-fed rats. Golgi membranes were isolated from cells as described in Methods and normalized by the total protein. EtOH sample was treated with CIP in the presence or absence of β-GP. ( g,h ) NMIIA-P-S1943 and Rab6a ( g ), and giantin and Rab6a ( h ) Western blot of the complexes pulled down with Rab6a Ab from the lysate of control VA-13 cells or cells treated with 35 mM EtOH for 72 h. Input was normalized by NMIIA-P-S1943 ( g ) or giantin ( h ). ( i ) Giantin Western blot of the lysates of VA-13 cells treated with scramble or GOLGB1 (giantin) siRNAs; β-actin was a loading control. ( j ) Rab6a and NMIIA-P-S1943 Western blot of the complexes pulled down with NMIIA-P-S1943 Ab VA-13 cells treated with scramble or GOLGB1 (giantin) siRNAs; input was normalized by Rab6a.

Article Snippet: MYH9 (myosin, heavy polypeptide 9, non-muscle, NMIIA), SAR1A and scrambled on-targetplus smartpool siRNAs were purchased from Santa Cruz Biotechnology.

Techniques: Immunostaining, Control, Isolation, Imaging, Fluorescence, Transfection, Plasmid Preparation, Western Blot

Journal: Scientific reports

Article Title: CREB3L2-mediated expression of Sec23A/Sec24D is involved in hepatic stellate cell activation through ER-Golgi transport.

doi: 10.1038/s41598-017-08703-6

Figure Lengend Snippet: Figure 4. COPII-mediated ER to Golgi transport is required for activation of LX-2 cells upon TGF- β1 stimulation. (a) LX-2 cells transfected with the indicated siRNA(s) were cultured for 24 h in DMEM supplemented with 10% FBS. After starvation for 24 h with DMEM supplemented with 0.5% FBS, the cells were untreated or treated with 1 ng/ml TGF-β1 and cultured for 3 days. Proteins were extracted and subjected to SDS-PAGE, followed by western blotting with anti-collagen I, anti-Sec23A, anti-Sec24D, anti-α-SMA and anti-GAPDH antibodies (lysates). Medium was collected for SDS-PAGE followed by western blotting with anti-collagen I antibody (medium). Shown is a representative immunoblot analysis (n = 3). (b and c) LX-2 cells transfected with the indicated siRNA(s) were cultured for 24 h in DMEM supplemented with 10% FBS. After starvation for 24 h with DMEM supplemented with 0.5% FBS, the cells were untreated or treated with 1 ng/ml TGF-β1 and cultured for 3 days. Proteins were extracted and subjected to SDS-PAGE followed by western blotting with anti-Sar1A, anti-Sar1B, anti-α-SMA and anti-GAPDH antibodies. (b) Representative immunoblots. (c) Quantification of immunoblots (n = 3). The band intensities of α-SMA were normalized

Article Snippet: Other antibodies were purchased from following companies: α-SMA (Abcam), CREB3L2/BBF2H7 (Atlas Antibodies), Sar1A (Novus Biological), β-actin (Chemicon), GAPDH (Chemicon), α-SMA-FITC antibody (SIGMA), Sar1B (Abnova), Smad2 (Cell signaling), pSmad2 (Cell signaling).

Techniques: Activation Assay, Transfection, Cell Culture, SDS Page, Western Blot

Journal: Neurobiology of disease

Article Title: Defective anterograde protein-trafficking contributes to endoplasmic reticulum-stress in a CLN1 disease model.

doi: 10.1016/j.nbd.2025.106890

Figure Lengend Snippet: Fig. 1. Impairment of COPII vesicle formation in CLN1 disease. (A) Graphical representation to demonstrate how the newly synthesized proteins from the ER to the Golgi are transported by COPII vesicles, while the transport from the Golgi to the ER is mediated by COPI vesicles. (B) Western blot analysis of Sar1 and COPII vesicle proteins (Sec23A, Sec24A, Sec31A and Sec13) using total homogenates of cortical tissues from 2-, 4- and 6-month-old WT and Cln1−/−mice (n = 4 animals in each group, p ≤0.05). (C) Western blot analysis of Sar1 and COPII vesicle proteins (Sec23A, Sec24A, Sec31A and Sec13) in the ER fractions from cortical tissues of 2-, 4- and 6-month-old WT and Cln1−/−mice (n = 4 animals in each group, p ≤0.05; NS = non-significant). Colocalization of ER-marker, calnexin, with Sar1 (D), Sec23A (E), Sec24A (F), Sec31A and Sec13 (G) in control and CLN1 patient fibroblasts was assessed using the Manders colocalization coefficient (n = 24 cells). The data are presented as the mean ± SD, and the ‘n’ numbers denote the number of biological replicates for each experiment. *p-values ≤0.05 were considered statistically significant and NS is non-significant. ER = endoplasmic reticulum; COPI = coat protein complex I; COPII = coat protein complex II; ERGIC = ER-Golgi intermediate compartment; ERES = Endoplasmic Reticulum Exit Sites.

Article Snippet: Myc-DDK tagged mouse Sar1 (OriGene Technologies Inc., Rockville, Cat#MR201967) and Mouse Flag-tagged CLN8 (Genecopoeia, Rockville, Cat# ExMm06485-M39) and PPT1 (Myc-DDK-tagged)-Human palmitoylprotein thioesterase-1 (OriGene Technologies Inc., Rockville, Cat# SKU RC203278) plasmids were used for the transfection studies.

Techniques: Synthesized, Western Blot, Marker, Control

Journal: Neurobiology of disease

Article Title: Defective anterograde protein-trafficking contributes to endoplasmic reticulum-stress in a CLN1 disease model.

doi: 10.1016/j.nbd.2025.106890

Figure Lengend Snippet: Fig. 2. Analysis of COPII vesicle proteins in the Golgi fractions from cortical tissues. (A) Western blot analysis of COPII vesicle proteins (Sar1, Sec23A, Sec24A, Sec31A and Sec13) in the Golgi fractions from cortical tissues of 2-, 4- and 6-month-old WT and Cln1−/−mice (n = 4 animals in each group, p ≤0.05; NS = non-significant). Colocalization of Golgi marker, GM-130, with COPII vesicle proteins: Sar1(B), Sec23A(C), Sec24A(D), Sec31A (E) and Sec13 (F) in control and CLN1 patient fibroblasts. Colocalization of Sar1, Sec23A, Sec24, Sec31A and Sec13 with the Golgi- marker, GM-130, was evaluated using the Manders’ colocalization coefficients (n = 24 cells). The data are presented as the mean ± SD and the “n”numbers denote the number of biological replicates for each experiment. *p-values ≤0.05 were considered statistically significant and NS were considered non-significant.

Article Snippet: Myc-DDK tagged mouse Sar1 (OriGene Technologies Inc., Rockville, Cat#MR201967) and Mouse Flag-tagged CLN8 (Genecopoeia, Rockville, Cat# ExMm06485-M39) and PPT1 (Myc-DDK-tagged)-Human palmitoylprotein thioesterase-1 (OriGene Technologies Inc., Rockville, Cat# SKU RC203278) plasmids were used for the transfection studies.

Techniques: Western Blot, Marker, Control

Journal: Neurobiology of disease

Article Title: Defective anterograde protein-trafficking contributes to endoplasmic reticulum-stress in a CLN1 disease model.

doi: 10.1016/j.nbd.2025.106890

Figure Lengend Snippet: Fig. 3. Ppt1-deficiency impairs interaction of Sar1 with inner layers of COPII vesicles. (A)Western blot analysis of Sec12 in the total homogenates of cortical tissues from 2-, 4- and 6-month-old WT and Cln1−/−mice (n = 4 animals in each group, p ≤0.05; NS = non-significant). (B) Western blot analysis of Sec12 in the ER fraction isolated from the cortical tissues of 2-, 4- and 6-month-old WT and Cln1−/−mice (n = 4). (C) Pull down experiments using anti-Sar1, which pulled down Sec12 from total homogenates of cortical tissues from 6-month-old WT and Cln1−/−mice (n = 4). (D) Anti-Sec12 pulls down Sar1 from total homogenates of cortical tissues from 6-month-old WT and Cln1 −/−mice (n = 4); (E) Colocalization of Sec12 with Sar1 in control and CLN1 patient fibroblasts; Colocalization between Sec12 and Sar1 was assessed using the Manders colocalization coefficients (n = 24 cells); (F) Anti-Sec23A pulls down Sar1 in total homogenates of cortical lysates from 6 month old WT and Cln1−/−mice (n = 4); (G) Anti-Sar1 pulls down Sec23A in total cortical lysates from 6 month old WT and Cln1−/−mice (n = 4); (H) Antibody to Sec24A pulls down Sar1 from total cortical lysates from 6 month old WT and Cln1 −/−mice (n = 4); (I) Antibody to Sar1 pulls down Sec24A from total cortical lysates from 6 month old WT and Cln1 −/−mice (n = 4). The data are presented as the mean ± SD and the ‘n’ numbers denote the number of biological replicates for each experiment. *p-values ≤0.05 were considered statistically significant.

Article Snippet: Myc-DDK tagged mouse Sar1 (OriGene Technologies Inc., Rockville, Cat#MR201967) and Mouse Flag-tagged CLN8 (Genecopoeia, Rockville, Cat# ExMm06485-M39) and PPT1 (Myc-DDK-tagged)-Human palmitoylprotein thioesterase-1 (OriGene Technologies Inc., Rockville, Cat# SKU RC203278) plasmids were used for the transfection studies.

Techniques: Western Blot, Isolation, Control

Journal: Neurobiology of disease

Article Title: Defective anterograde protein-trafficking contributes to endoplasmic reticulum-stress in a CLN1 disease model.

doi: 10.1016/j.nbd.2025.106890

Figure Lengend Snippet: Fig. 4. S-Palmitoylation of COPII vesicle- and Cln8-proteins. (A)Acyl-Rac assay to determine S-palmitoylation of endogenous Sar1 in WT mice (n = 4). (B) S-palmitoylation of endogenous Sec23A in WT mice (n = 4). (C) S- palmitoylation of endogenous Sec24A in WT mice (n = 4). (D) S-palmitoylation of endogenous Sec31A in WT mice (n = 4). (E) S-palmitoylation of endogenous Sec13 in WT mice (n = 4). (F) Level of S-palmitoylated endogenous Sar1 in cortical tissues from 6-month-old WT and Cln1−/−mice (n = 4). (G) Acyl Rac assay to detect S- palmitoylation of Sar1 expressed in HEK293T cells co-transfected with Myc-Sar1 construct. (H) CSS-palm analysis of mouse Sar1 (NCBI GenBank: EDL32136.1) predicting that Cys102 and 178 are the S-palmitoylated residues. The predicted cysteine residues are marked in red. (I) Acyl-Rac assay using transfected HEK293T cells with mutant Sar1 (Cys102Ala) and (Cys178Ala) constructs to confirm that Cys102 is the palmitoylation site in Sar1; (PBS-B: Negative Control, PBS-UB: PBS unbound non-S-Plamitoylated protein, HA-B: Hydroxylamine bound S-palmitoylated protein, HA-UB: Hydroxylamine unbound non-S-palmitoylated protein (see methods for details). The data are presented as the mean ± SD and the ‘n’ numbers denote the number of biological replicates for each experiment. *p-values ≤0.05 were considered statistically significant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Myc-DDK tagged mouse Sar1 (OriGene Technologies Inc., Rockville, Cat#MR201967) and Mouse Flag-tagged CLN8 (Genecopoeia, Rockville, Cat# ExMm06485-M39) and PPT1 (Myc-DDK-tagged)-Human palmitoylprotein thioesterase-1 (OriGene Technologies Inc., Rockville, Cat# SKU RC203278) plasmids were used for the transfection studies.

Techniques: Transfection, Construct, Mutagenesis, Negative Control

Journal: Orphanet Journal of Rare Diseases

Article Title: Molecular analysis and intestinal expression of SAR1 genes and proteins in Anderson's disease (Chylomicron retention disease)

doi: 10.1186/1750-1172-6-1

Figure Lengend Snippet: The expression of the SAR1A and SAR1B genes in intestinal biopsies of healthy individuals and an Anderson's disease patient . The graph shows the log base 2 fold change (increase or decrease), as measured by RTQPCR, in the expression of the SAR1A and SAR1B genes in 4 healthy normal adults (NA1, NA2 NA3 and NA4), one healthy young adult (YA1) and two healthy children (C1 and C2) normalized to an Anderson's disease patient (AD3). Gene expression was measured in biopsies obtained on two separate occasions from the young adult (YA1) and from one of the children (C1). The standard deviation of the measurements are shown and, except for the value for SAR1A for NA1, all the differences are significant (p < 0.01) as compared to the patient.

Article Snippet: Real time quantitative PCR was performed with the ABI Prism model 7300 Sequence Detection System (Applied Biosystems), using the Taqman MGB specific probes for SAR1A (Hs00833068_s1), for SAR1B (Hs01011583_m1) (Applied Biosystems), and Absolute blue QPCR ROX mix (Abgene).

Techniques: Expressing, Gene Expression, Standard Deviation