rabbit anti stx17 polyclonal antibody  (Proteintech)

Journal: Nature Communications

Article Title: CHIP ameliorates nonalcoholic fatty liver disease via promoting K63- and K27-linked STX17 ubiquitination to facilitate autophagosome-lysosome fusion

doi: 10.1038/s41467-024-53002-0

Figure Lengend Snippet: a Eight-week-old WT and H-KO mice individually housed were fed a HFD for 20 weeks. b Representative livers of mice fed a HFD (left), and the calculated percentage of liver weight/body weight ratio (right). Grid cubes are 5 mm*5 mm. c Representative western blot images exhibiting P62, LC3, and STX17 expression in the liver samples of HFD-fed mice. d – g Representative images of liver samples stained with H&E (n = 13), Oil red O (WT, n = 7; H-KO, n = 9), f4/80 immunohistochemistry (n = 7), and Sirius red (n = 12). h – i Hepatic TG and TC levels, and serum ALT and AST levels in mice fed an HFD (n = 7). j Glucose tolerance tests (n = 5) and k insulin tolerance tests (n = 5) were conducted. l Representative western blot images demonstrating the insulin-stimulated phosphorylation of AKT in livers. Mice were injected with either PBS (−) or insulin (+). The error bars in ( j ) and ( k ) indicate SD. Data are presented as individual data points, mean ± SD is assessed by unpaired two-tailed t -test.

Article Snippet: Subsequent to blocking the non-specific interactions, the sections were incubated with anti-CHIP rabbit polyclonal antibodies (HPA041222; dilution 1:2000; Millipore Sigma) or anti-STX17 rabbit polyclonal antibodies (PA5-40127; dilution 1:4000; Invitrogen) for 30 min at room temperature.

Techniques: Western Blot, Expressing, Staining, Immunohistochemistry, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: CHIP ameliorates nonalcoholic fatty liver disease via promoting K63- and K27-linked STX17 ubiquitination to facilitate autophagosome-lysosome fusion

doi: 10.1038/s41467-024-53002-0

Figure Lengend Snippet: a Eight-week-old WT and H-KO mice individually housed were fed a HFHFD for 24 weeks. b Representative livers of mice fed a HFHFD (left), and the calculated percentage of liver weight/body weight ratio (right) (n = 9). Grid cubes are 5 mm*5 mm. c , d Representative western blot images and quantitation graph exhibiting P62, LC3, and STX17 expression in the liver samples of HFHFD-fed mice (n = 6). e – h Representative images of liver samples (n = 7) stained with H&E, Oil red O, f4/80 immunohistochemistry, and Sirius red. i , j Hepatic TG (n = 7) and TC (WT, n = 7; H-KO, n = 8) levels, and serum ALT (n = 7) and AST (n = 8) levels in mice fed a HFHFD. k , l qRT-PCR was used to quantify the relative transcript levels of inflammation and fibrosis genes in mouse liver samples (n = 7–9). Data are presented as individual data points, mean ± SD is assessed by unpaired two-tailed t -test. GOI, gene of interest.

Article Snippet: Subsequent to blocking the non-specific interactions, the sections were incubated with anti-CHIP rabbit polyclonal antibodies (HPA041222; dilution 1:2000; Millipore Sigma) or anti-STX17 rabbit polyclonal antibodies (PA5-40127; dilution 1:4000; Invitrogen) for 30 min at room temperature.

Techniques: Western Blot, Quantitation Assay, Expressing, Staining, Immunohistochemistry, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: CHIP ameliorates nonalcoholic fatty liver disease via promoting K63- and K27-linked STX17 ubiquitination to facilitate autophagosome-lysosome fusion

doi: 10.1038/s41467-024-53002-0

Figure Lengend Snippet: a Representative immunohistochemical staining images for CHIP and STX17 in formalin-fixed paraffin-embedded (FFPE) liver tissues. H&E staining of FFPE human liver samples diagnosed as normal and NASH are shown at top of the image. Scale bar shown is 100 µm. b , c Mice were fed a NCD, HFD, or HFHFD for 20 weeks (n = 5). Western blotting and quantification of the liver samples. d Lysates of HepG2 cells transfected with the plasmid expressing HA-VAMP8 were immunoprecipitated using anti-STX17 antibodies, followed by western blotting. e The plasmid expressing FLAG-CHIP was co-transfected with the plasmid expressing HA-STX17, HA-SNAP29, or HA-VAMP8 in 293FT cells. The lysates were immunoprecipitated using anti-HA antibodies, followed by western blotting. f HepG2 cells expressing HA-VAMP were treated with OA. The lysates were immunoprecipitated with anti-CHIP antibodies, followed by western blotting. Lysates from each cell type treated with OA overnight were immunoprecipitated with anti-STX17 antibodies, followed by western blotting. g HepG2 cells stably transfected with plasmids #3 or #4 expressing CHIP-shRNAs. h Primary hepatocytes isolated from WT and H-KO mice. i HepG2 cells transfected with plasmids overexpressing HA-CHIP-WT, HA-CHIP-H260Q, or HA-CHIP-K30A. Immunofluorescence analyses of HepG2 cells using confocal microscopy was conducted as follows: j HepG2 cells; k HepG2 cells were transfected with the plasmid expressing HA-CHIP and FLAG-STX17; l CHIP was knocked down in HepG2 cells, followed by transfection with plasmids expressing FLAG-STX17 and HA-VAMP. m HepG2 cells stably overexpressing CHIP-WT, CHIP-H260Q, or CHIP-K30A were transfected with plasmids overexpressing FLAG-STX17 and HA-VAMP8. HepG2 cells overexpressing indicated plasmids were treated with OA overnight, followed by immunofluorescent analysis employing anti-CHIP, anti-STX17, anti-HA, and anti-FLAG antibodies. The graphs indicate fluorescence intensities (white arrow). Data are presented as individual data points, mean ± SD is assessed by unpaired two-tailed t -test.

Article Snippet: Subsequent to blocking the non-specific interactions, the sections were incubated with anti-CHIP rabbit polyclonal antibodies (HPA041222; dilution 1:2000; Millipore Sigma) or anti-STX17 rabbit polyclonal antibodies (PA5-40127; dilution 1:4000; Invitrogen) for 30 min at room temperature.

Techniques: Immunohistochemical staining, Staining, Formalin-fixed Paraffin-Embedded, Western Blot, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Stable Transfection, Isolation, Immunofluorescence, Confocal Microscopy, Fluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: CHIP ameliorates nonalcoholic fatty liver disease via promoting K63- and K27-linked STX17 ubiquitination to facilitate autophagosome-lysosome fusion

doi: 10.1038/s41467-024-53002-0

Figure Lengend Snippet: a Lysates of HepG2 cells treated with or without OA were immunoprecipitated using anti-STX17 antibodies, followed by western blotting. b , c HepG2 cells stably transfected with HA-Ub, and plasmid #3 or #4 expressing CHIP-shRNA, or plasmids overexpressing FLAG-CHIP-WT, FLAG-CHIP-HQ, or FLAG-CHIP-KA, as indicated, were treated with OA overnight. Lysates were immunoprecipitated using anti-STX17 antibodies, followed by western blotting. d , e 293FT cells were transfected with plasmids expressing FLAG-STX17-WT, FLAG-STX17-1KR, FLAG-STX17-3KR, FLAG-STX17-4KR, myc-CHIP, HA-Ub, or HA-VAMP8, as indicated. Cell lysates were immunoprecipitated with anti-FLAG antibodies, followed by western blotting. f Immunofluorescence analysis of HepG2 cells was conducted by confocal microscopy, as follows. Cells were treated with OA overnight and transfected with plasmids expressing FLAG-STX17-WT, FLAG-STX17-1KR, FLAG-STX17-3KR, FLAG-STX17-4KR, and HA-VAMP8, as indicated, followed by immunofluorescence analysis using anti-FLAG and anti-HA antibodies. Graphs indicate the fluorescence intensity (white arrow). g , h 293FT cells were transfected with plasmids expressing HA-Ub-WT, HA-Ub-K63, HA-Ub-K27, HA-Ub-K63K27, HA-UbK63RK27R, FLAG-STX17, or myc-CHIP, as indicated. Cell lysates were immunoprecipitated with anti-FLAG antibodies, followed by western blotting. Lysates of ( i ) primary hepatocytes isolated from WT and H-KO mice, and ( j ) NCD-, HFD-, and HFHFD-fed mouse liver samples immunoprecipitated using anti-STX17 antibodies, followed by western blotting (n = 4). Data are presented as individual data points, mean ± SD is assessed by paired two-tailed t -test.

Article Snippet: Subsequent to blocking the non-specific interactions, the sections were incubated with anti-CHIP rabbit polyclonal antibodies (HPA041222; dilution 1:2000; Millipore Sigma) or anti-STX17 rabbit polyclonal antibodies (PA5-40127; dilution 1:4000; Invitrogen) for 30 min at room temperature.

Techniques: Immunoprecipitation, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Expressing, shRNA, Immunofluorescence, Confocal Microscopy, Fluorescence, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: CHIP ameliorates nonalcoholic fatty liver disease via promoting K63- and K27-linked STX17 ubiquitination to facilitate autophagosome-lysosome fusion

doi: 10.1038/s41467-024-53002-0

Figure Lengend Snippet: Representative images with autophagosome (yellow puncta) and autolysosome (red puncta). a HepG2 cells with STX17 knockdown or ( b ) stable overexpression of STX17-WT, STX17-1KR, STX17-3KR or STX17-4KR were transfected with RFP-GFP-LC3 and treated with OA overnight. Representative images demonstrating lipid staining with Nile red (orange) and Hoechst staining of DNA (blue). c HepG2 cells stably overexpressing CHIP-WT were co-transfected with plasmids #2 or #4 expressing STX17-shRNAs, and ( d ) HepG2 cells overexpressing STX17-WT were co-transfected with plasmids #3 or #4 expressing CHIP-shRNAs. Both cell lines were treated with OA overnight. Eight-week-old mice were individually housed for 10 weeks with a HFD and intravenously injected with the plasmids expressing control (CON), HA-mSTX17-WT (mSTX), or HA-mSTX17-1KR (m1KR) on days 4 and 1 prior to sacrifice. e Representative livers of mice fed a HFD (left) and the calculated percentage of liver weight/body weight ratio (right) (n = 7). Grid cubes are 5 mm*5 mm. f , g Representative images of liver samples stained with H&E or Oil red O (n = 8). h Hepatic TG (n = 9) and TC (n = 7) levels in mice fed an HFD. i Calculated number of autophagic vesicles per field (n = 4). j Representative TEM images (n = 4). Autophagosomes (yellow arrows) and autolysosomes (blue arrows) are indicated in the images. k Lysates of mSTX17 and m1KR liver samples were immunoprecipitated with anti-STX17 antibodies, followed by western blotting. l Immunofluorescence analyses of CON, mSTX17, and m1KR mouse liver samples using confocal microscopy were conducted using anti-STX17, anti-SNAP29, and anti-VAMP8 antibodies. Graphs indicate fluorescence intensities (white arrow). Data are presented as individual data points, mean ± SD is assessed by unpaired two-tailed t -test. LD lipid droplet.

Article Snippet: Subsequent to blocking the non-specific interactions, the sections were incubated with anti-CHIP rabbit polyclonal antibodies (HPA041222; dilution 1:2000; Millipore Sigma) or anti-STX17 rabbit polyclonal antibodies (PA5-40127; dilution 1:4000; Invitrogen) for 30 min at room temperature.

Techniques: Knockdown, Over Expression, Transfection, Staining, Stable Transfection, Expressing, Injection, Control, Immunoprecipitation, Western Blot, Immunofluorescence, Confocal Microscopy, Fluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: CHIP ameliorates nonalcoholic fatty liver disease via promoting K63- and K27-linked STX17 ubiquitination to facilitate autophagosome-lysosome fusion

doi: 10.1038/s41467-024-53002-0

Figure Lengend Snippet: a – i ; r , s Eight-week-old mice were individually housed for 16 weeks with a HFD and intravenously injected with AAV8-GFP or AAV8-CHIP-GFP 8 weeks prior to sacrifice. a Representative livers of mice fed a HFD (left) and the calculated percentage of liver weight/body weight ratio (right) (n = 12). Grid cubes are 5 mm*5 mm. b – d Representative images of liver samples stained with Oil red O, f4/80 immunohistochemistry, and Sirius red. e , f Hepatic TG (n = 11), TC levels (n = 11), and serum ALT (n = 10), AST (n = 8) levels in mice fed an HFD. g Glucose tolerance tests (n = 8) and h insulin tolerance tests (n = 7) were conducted. (i) Representative western blot images demonstrating the insulin-stimulated phosphorylation of AKT in livers. Mice were injected with either PBS (−) or insulin (+). j , q Eight-week-old mice were individually housed for 20 weeks with a HFHFD and intravenously injected with AAV8-GFP or AAV8-CHIP-GFP 10 weeks prior to sacrifice. j Representative livers of mice fed a HFHFD (left) and the calculated percentage of liver weight/body weight ratio (right) (n = 7). Grid cubes are 5 mm*5 mm. k – m Representative images of liver samples stained with Oil red O, f4/80 immunohistochemistry, and Sirius red. n , o qRT-PCR was used to quantify the relative transcript levels of inflammation and fibrosis genes in mouse liver samples (n = 7). p , q Hepatic TG (n = 7), TC (n = 7) levels, and serum ALT (n = 8), AST (n = 8) levels in mice fed an HFD. r Lysates of HFD-fed CON and OE mouse liver samples were immunoprecipitated with anti-STX17, followed by western blotting. s Representative images of immunofluorescence analyses of HFD-fed CON and OE mouse liver samples using confocal microscopy conducted using anti-STX17, anti-SNAP29, and anti-VAMP8 antibodies. Graphs indicate fluorescence intensities (white arrow). The error bars in ( g ) and ( h ) indicate SD. Data are presented as individual data points, mean ± SD is assessed by unpaired two-tailed t -test. GOI gene of interest.

Article Snippet: Subsequent to blocking the non-specific interactions, the sections were incubated with anti-CHIP rabbit polyclonal antibodies (HPA041222; dilution 1:2000; Millipore Sigma) or anti-STX17 rabbit polyclonal antibodies (PA5-40127; dilution 1:4000; Invitrogen) for 30 min at room temperature.

Techniques: Injection, Staining, Immunohistochemistry, Western Blot, Quantitative RT-PCR, Immunoprecipitation, Immunofluorescence, Confocal Microscopy, Fluorescence, Two Tailed Test