sc398214 af6242 af3242 af6261  (Santa Cruz Biotechnology)

Journal: Inflammation

Article Title: A Novel Insight into Chronic Pancreatitis Pathogenesis: the USP1/ITGB5 Axis-Mediated Stellate Cell Activation

doi: 10.1007/s10753-025-02434-x

Figure Lengend Snippet: Proteomics reveals impairment of ECM degradation in PSCs with USP1 knockdown. A , B . USP1 expression was estimated in PSCs treated with different concentration of TGF-β1 (n = 3). C , D . The USP1 expression after lentiviral transfection was detected by real-time PCR ( C ) and western blot ( D ) ( n = 3). E Heap map of differentially expressed proteins (DEPs) in label-free proteomic ( n = 4). F . GO enrichment analysis of DEPs associated with collagen fibers. G . Heat map of collagen protein (n = 4). H . The protein expression of COL1A1, COL1A2, and FN in the PSCs was determined by western blot ( n = 3). I . Immunofluorescence staining of COL1A1 in the PSCs ( n = 3). Bar: 50 μm. J . Immunofluorescence staining of α-SMA. Bar: 50 μm. K . Quantitative analysis of the fluorescence intensity of COL1A1 ( I ) and α-SMA (J) ( n = 3). **, p < 0.01; ***, p < 0.001

Article Snippet: COL1A2 ITGB5 PI3K p-PI3K AKT , 343,277 SC398214 AF6242 AF3242 AF6261 , 1: 500 1:1000 1:1000 1:1000 1:1000 , Zenbio Santa Cruz Affinity Affinity Affinity , WB WB/IP WB WB WB , .

Techniques: Knockdown, Expressing, Concentration Assay, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: Inflammation

Article Title: A Novel Insight into Chronic Pancreatitis Pathogenesis: the USP1/ITGB5 Axis-Mediated Stellate Cell Activation

doi: 10.1007/s10753-025-02434-x

Figure Lengend Snippet: USP1 knockdown promotes ITGB5 ubiquitination-mediated degradation. ( A ) Venn plot showing a total of 2015 proteins bind to USP1 under TGF-β1 stimulation. (B) KEGG pathway analysis and GO enrichment analysis pair of 2015 proteins in ( A ). ( C ) Label-free proteomics combined with IP-LC/MS to analysis the USP1 downstream target. The intersection of differential downregulated expressed protein of label-free proteomics, IP-LC/MS, and the upregulated genes of GSE41418 dataset. ( D ) The ITGB5-associated PPI was established via the String database. E-F. The expression of ITGB5 of the PSCs (E) ( n = 3) and pancreatic tissues ( F ) ( n = 6) was estimated by western blot. G . Co-IP shows the interaction of USP1 and ITGB5 in the PSCs ( n = 3). H . Ubiquitination detection of ITGB5 in the PSCs were measured by Co-IP ( n = 3). **, p < 0.01; ***, p < 0.001.

Article Snippet: COL1A2 ITGB5 PI3K p-PI3K AKT , 343,277 SC398214 AF6242 AF3242 AF6261 , 1: 500 1:1000 1:1000 1:1000 1:1000 , Zenbio Santa Cruz Affinity Affinity Affinity , WB WB/IP WB WB WB , .

Techniques: Knockdown, Ubiquitin Proteomics, Liquid Chromatography with Mass Spectroscopy, Expressing, Western Blot, Co-Immunoprecipitation Assay

Journal: Inflammation

Article Title: A Novel Insight into Chronic Pancreatitis Pathogenesis: the USP1/ITGB5 Axis-Mediated Stellate Cell Activation

doi: 10.1007/s10753-025-02434-x

Figure Lengend Snippet: ITGB5 knockdown inhibits PSC activation by inhibiting the PI3K/AKT pathway. A . The ITGB5 expression in the PSCs after lentiviral transfection was detected by western blot ( n = 3). B , C . The expression of COL1A1 ( B ) and FN ( C ) was detected by western blot ( n = 3). D . IF staining showed the α-SMA expression ( n = 3). Bar: 50 μm. E . KEGG analysis of DEPs of label-free proteomics. F , G . The phosphorylation levels of PI3K and AKT and the PI3K and AKT protein expression was measured by western blot (n = 3). H , I . The expression of COL1A1 and FN was detected by western blot ( n = 3). J . α-SMA expression in the PSCs was determined by IF staining (n = 3). Bar: 50 μm. **, p < 0.01; ***, p < 0.001.

Article Snippet: COL1A2 ITGB5 PI3K p-PI3K AKT , 343,277 SC398214 AF6242 AF3242 AF6261 , 1: 500 1:1000 1:1000 1:1000 1:1000 , Zenbio Santa Cruz Affinity Affinity Affinity , WB WB/IP WB WB WB , .

Techniques: Knockdown, Activation Assay, Expressing, Transfection, Western Blot, Staining, Phospho-proteomics

Journal: Inflammation

Article Title: A Novel Insight into Chronic Pancreatitis Pathogenesis: the USP1/ITGB5 Axis-Mediated Stellate Cell Activation

doi: 10.1007/s10753-025-02434-x

Figure Lengend Snippet: ITGB5 overexpression increased extracellular matrix. ( A ) ITGB5 expression in the PSCs was detected by western blot ( n = 3). ( B ) COL1A1 expression in the PSCs was detected by western blot ( n = 3). (C) FN expression in the PSCs was detected by western blot ( n = 3). ( D ) Immunofluorescence staining of α-SMA in the PSCs ( n = 3). Bar: 50 μm. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: COL1A2 ITGB5 PI3K p-PI3K AKT , 343,277 SC398214 AF6242 AF3242 AF6261 , 1: 500 1:1000 1:1000 1:1000 1:1000 , Zenbio Santa Cruz Affinity Affinity Affinity , WB WB/IP WB WB WB , .

Techniques: Over Expression, Expressing, Western Blot, Immunofluorescence, Staining

Journal: Inflammation

Article Title: A Novel Insight into Chronic Pancreatitis Pathogenesis: the USP1/ITGB5 Axis-Mediated Stellate Cell Activation

doi: 10.1007/s10753-025-02434-x

Figure Lengend Snippet: Graphical summary of the possible mechanisms of USP1/ITGB5 axis in CP progression. USP1 deubiquitinates and stabilizes ITGB5, upregulates ITGB5 expression, and ITGB5 activated the PI3K/AKT pathway and subsequently activates PSCs, leading to pancreatic fibrotic injury in CP. Arrow: promotion; “tee” head: inhibition

Article Snippet: COL1A2 ITGB5 PI3K p-PI3K AKT , 343,277 SC398214 AF6242 AF3242 AF6261 , 1: 500 1:1000 1:1000 1:1000 1:1000 , Zenbio Santa Cruz Affinity Affinity Affinity , WB WB/IP WB WB WB , .

Techniques: Expressing, Inhibition

Journal: bioRxiv

Article Title: Basement membrane mechanics drives patterned response to developmental signalling

doi: 10.64898/2026.02.13.705301

Figure Lengend Snippet: a. Principal component analysis of bulk RNA sequencing data from hPSCs on stiff and soft substrates after 4 days of culture, showing three biological replicates projected onto the first two principal components. b. Representative immunofluorescence images (maximum projection) of the colony bottom (bottom 5 confocal planes) of hPSCs on stiff and soft substrates, stained for integrin β5. Scale bar = 50 μm; zoomed in images scale bar = 20 μm. Individual data came from N = 3 independent experiments representing biological replicates. c. Representative immunofluorescence images (maximum projection) of colony bottom (bottom 5 confocal planes) of hPSCs on stiff and soft substrates, stained for pFAK and F-Actin. Scale bar = 50 μm; zoomed in images scale bar = 20 μm. Individual data came from N = 3 independent experiments representing biological replicates. d. Left: Schematic representing the experimental protocol involving FAK inhibition (FAKi) before the BMP4 treatment of hPSCs on stiff and soft substrates. Right: Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff and soft substrates stained for pSMAD1/5 and DAPI after 1 h of BMP4 treatment, with or without the FAKi treatment. Scale bar = 50 μm. e. Quantification of pSMAD1/5 signal from immunofluorescence images of hPSCs in stiff control, soft control and stiff FAK inhibitor (FAKi) condition, after 1 h of BMP4 treatment. The signal intensity in each nucleus is plotted as a function of the distance of that nucleus from the colony edge, normalized by the 98 th percentile value of each image. The continuous line represents the mean signal intensity and edges of the shaded areas represent +/- standard deviation from N = 3 biological replicates. f. Left: Schematic representing the experimental protocol involving PI3K inhibition (PI3Ki) before the BMP4 treatment of hPSCs on stiff and soft substrates. Right: Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff and soft substrates stained for pSMAD1/5 and DAPI after 1 h of BMP4 treatment, with or without the PI3Ki treatment. Scale bar = 50 μm. g. Quantification of pSMAD1/5 signal from immunofluorescence images of hPSCs in stiff control, soft control and stiff PI3K inhibitor (PI3Ki) condition, after 1 h of BMP4 treatment. The signal intensity in each nucleus is plotted as a function of the distance of that nucleus from the colony edge, normalized by the 98 th percentile value of each image. The continuous line represents the mean signal intensity and edges of the shaded areas represent +/- standard deviation from N = 3 biological replicates.

Article Snippet: The following primary antibodies were used: Brachyury (1:500; Cell Signalling Technology, #81694S), integrin β5 (1:200; Cell Signalling Technology, #3629S), pFAK (1:200; Thermo Scientific, #44-624G), active integrin β1 (1:100; Abcam, #ab30394), ZO-1 (1:250; Thermo Scientific, #33-9100), BMPR1A (1:50; Santa Cruz Biotechnology, #sc-518037), Myosin Regulatory Light Chain 2 (1:100; Cell Signalling Technology, #3672S), Claudin-6 (1:100; Santa Cruz Biotechnology, #sc-17669), laminin (1:200; Thermo Scientific, #PA1-16730).

Techniques: RNA Sequencing, Immunofluorescence, Staining, Inhibition, Control, Standard Deviation

Journal: bioRxiv

Article Title: Basement membrane mechanics drives patterned response to developmental signalling

doi: 10.64898/2026.02.13.705301

Figure Lengend Snippet: a. Volcano plot of DEGs showing the log2 fold change (x axis) of hPSCs on soft substrates relative to stiff substrates as a function of the -log10(adjusted p value) (y axis). In blue are genes that are significantly upregulated on soft substrates at a p-adjusted value of 0.05. In red are genes that are significantly downregulated on soft substrates at a p-adjusted value of 0.05. The total number of significantly differentially expressed genes equals 62. Data was generated from N = 3 independent experiments representing biological replicates. b. Dot plot of enriched GO terms for the soft control vs stiff control comparison. The top 5 GO biological processes with the largest gene ratios are shown. The size of the dots represents the number of genes in the significant DEG list associated with the GO term and the colour represents the p-adjusted values. The plot is split by sign of the normalised enrichment score (NES, NES>0 as activated, NES<0 as suppressed). Data was generated from N = 3 independent experiments representing biological replicates. c. Normalised counts obtained from RNA-sequencing data of hPSCs on stiff and soft substrates for each integrin ( ITG ) subunit expressed. Differential expression analysis was performed using DESeq2 with the Wald test. Data was generated from N = 3 independent experiments representing biological replicates. d. Western Blot of pFAK and GAPDH in hPSCs on stiff and soft substrates without BMP4 treatment (control). e. Dot plot of enriched GO terms for the stiff FAK inhibitor vs stiff control comparison. The top 5 GO biological processes with the largest gene ratios are shown. The size of the dots represents the number of genes in the significant DEG list associated with the GO term and the colour represents the p-adjusted values. The plot is split by sign of the normalised enrichment score (NES, NES>0 as activated, NES<0 as suppressed). Data was generated from N = 3 independent experiments representing biological replicates. from N = 3 independent experiments representing biological replicates. f. Dot plot of enriched GO terms for the stiff PI3K inhibitor vs stiff control comparison. The top 5 GO biological processes with the largest gene ratios are shown. The size of the dots represents the number of genes in the significant DEG list associated with the GO term and the colour represents the p-adjusted values. The plot is split by sign of the normalised enrichment score (NES, NES>0 as activated, NES<0 as suppressed). Data was generated from N = 3 independent experiments representing biological replicates. g. Left: Schematic representing the experimental protocol involving PTEN inhibition (PTENi) before BMP4 treatment. Right: Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff and soft substrates, treated with BMP4 for 1 h, with or without prior PTEN inhibition, stained for pSMAD1/5 and DAPI. Scale bar = 50 μm. h. Quantification of pSMAD1/5 signal from immunofluorescence images of hPSCs in stiff and soft control, and stiff and soft PTENi condition, after 1 h of BMP4 treatment. The signal intensity in each nucleus is plotted as a function of the distance of that nucleus from the colony edge, normalized by the 98 th percentile value of each image. The continuous line represents the mean signal intensity and edges of the shaded areas represent +/- standard deviation from N = 3 biological replicates.

Article Snippet: The following primary antibodies were used: Brachyury (1:500; Cell Signalling Technology, #81694S), integrin β5 (1:200; Cell Signalling Technology, #3629S), pFAK (1:200; Thermo Scientific, #44-624G), active integrin β1 (1:100; Abcam, #ab30394), ZO-1 (1:250; Thermo Scientific, #33-9100), BMPR1A (1:50; Santa Cruz Biotechnology, #sc-518037), Myosin Regulatory Light Chain 2 (1:100; Cell Signalling Technology, #3672S), Claudin-6 (1:100; Santa Cruz Biotechnology, #sc-17669), laminin (1:200; Thermo Scientific, #PA1-16730).

Techniques: Generated, Control, Comparison, RNA Sequencing, Quantitative Proteomics, Western Blot, Inhibition, Immunofluorescence, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Basement membrane mechanics drives patterned response to developmental signalling

doi: 10.64898/2026.02.13.705301

Figure Lengend Snippet: a. Top: Schematic showing the part of the hPSC colonies that was imaged using confocal microscopy (red box). Bottom: Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff and soft substrates, stained for BMPR1A and DAPI. Images were reconstructed in x-z plane (along the apicobasal axis); 50 imaging planes were projected in y-axis. Scale bar = 15 μm. b. Top: Schematic showing the part of the hPSC colonies that was imaged using structured illumination microscopy (SIM). Bottom: Representative immunofluorescence images (maximum projection) of hPSCs on stiff and soft substrates, stained for BMPR1A and with CellMask HCS dye staining the cytoplasm. Images were reconstructed in x-z (i.e., along the apicobasal axis), and the top 10 μm of the apicobasal axis were imaged; 40 imaging planes were projected in y-axis. Scale bar = 10 μm. c. Plot of the fraction of BMPR1A-positive pixels against the distance from cell surface from SIM images. The continuous line represents the mean signal intensity and edges of the shaded areas represent +/- standard deviation from N = 3 biological replicates. d. Schematic of apical and basolateral membrane domains in epithelial hPSCs, defined by the localisation of phospholipids PIP2 and PIP3. Schematic also represents the hypothesis of polarity disruption as a consequence of substrate stiffness change. e. Representative images of hPSCs on stiff and soft substrates in control conditions and upon FAK inhibitor (FAKi) or PI3K inhibitor (PI3Ki) treatment, transfected with the plasmid encoding the GFP-tagged PH domain of Akt. Images were reconstructed in the x-z plane, i.e., along the apicobasal axis. Scale bar = 20 μm. f. Quantification of the apical/basolateral ratio of PH Akt-GFP (labelling PIP3) signal intensity in individual hPSCs on stiff and soft substrates in control condition as well as on stiff substrates upon FAK inhibitor (FAKi) or PI3K inhibitor (PI3Ki) treatment. See for quantification pipeline. Individual data came from N = 3 independent experiments representing biological replicates. P-value determined by Welch’s t-test. **** indicates p-value < 0.0001. g. Top: Schematic showing the part of the hPSC colonies that was imaged using confocal microscopy (red box). Bottom: Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff and soft substrates, stained for the active form of integrin β1 and DAPI. Images were reconstructed in x-z plane (along the apicobasal axis); 50 imaging planes were projected in y-axis. Scale bar = 15 μm. h. Top: Schematic showing the part of the hPSC colonies that was imaged using structured illumination microscopy (SIM) (red box). Bottom: Representative immunofluorescence images (maximum projection) of hPSCs on stiff and soft substrates, stained for the active form of integrin β1 and with HCS dye staining the cytoplasm. Images were reconstructed in x-z (i.e., along the apicobasal axis), and the top 10 μm of the apicobasal axis were imaged; 20 imaging planes were projected in the y-axis. Scale bar = 10 μm. i. Plot of the fraction of active integrin β1-positive pixels against the distance from the cell surface from SIM images. The continuous line represents the mean signal intensity and edges of the shaded areas represent +/- standard deviation from N = 3 biological replicates.

Article Snippet: The following primary antibodies were used: Brachyury (1:500; Cell Signalling Technology, #81694S), integrin β5 (1:200; Cell Signalling Technology, #3629S), pFAK (1:200; Thermo Scientific, #44-624G), active integrin β1 (1:100; Abcam, #ab30394), ZO-1 (1:250; Thermo Scientific, #33-9100), BMPR1A (1:50; Santa Cruz Biotechnology, #sc-518037), Myosin Regulatory Light Chain 2 (1:100; Cell Signalling Technology, #3672S), Claudin-6 (1:100; Santa Cruz Biotechnology, #sc-17669), laminin (1:200; Thermo Scientific, #PA1-16730).

Techniques: Confocal Microscopy, Immunofluorescence, Staining, Imaging, Microscopy, Standard Deviation, Membrane, Disruption, Control, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Basement membrane mechanics drives patterned response to developmental signalling

doi: 10.64898/2026.02.13.705301

Figure Lengend Snippet: a. Representative immunofluorescence images of the colony apical surface of hPSCs on stiff and soft substrates, without permeabilization, stained for pan-laminin and DAPI. Scale bar = 50 μm. b. Quantification of apical laminin signal intensity across images. Individual data came from N = 3 independent experiments representing biological replicates. P-value determined by Welch’s t-test. *** indicates p-value < 0.001. c. Top: Schematic of the hypothesized apical cell surface in hPSCs on stiff and soft substrates. Green arrows point to microvilli, red rectangle represents the plane imaged with SEM. Bottom: SEM images of the apical surface of hPSCs on stiff and soft substrates. Green arrows point to individual microvilli. Scale bar = 10 μm; zoomed in images scale bar = 2 μm. Representative images came from N = 3 independent experiments representing biological replicates. d. Top: Schematic representing the experimental protocol involving soluble laminin addition into the cell medium of hPSCs on stiff substrates before the BMP4 treatment. Bottom: Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff substrates stained for pSMAD1/5 and DAPI after 1 h of BMP4 treatment, with or without addition of soluble laminin into the cell medium. Scale bar = 50 μm. e. Quantification of pSMAD1/5 signal from immunofluorescence images of hPSCs on stiff substrates, with or without the addition of soluble laminin into the cell medium and after 1 h of BMP4 treatment. The signal intensity in each nucleus is plotted as a function of the distance of that nucleus from the colony edge, normalized by the 98 th percentile value of each image. The continuous line represents the mean signal intensity and edges of the shaded areas represent +/- standard deviation from N = 3 biological replicates. f. Top: Schematic showing the part of the hPSC colonies that was imaged using confocal microscopy (red box). Bottom: Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff substrates, with or without the addition of soluble laminin, stained for the active form of integrin β1 and DAPI. Images were reconstructed in the x-z plane (along the apicobasal axis); 50 imaging planes were projected in y-axis. Scale bar = 20 μm. g. Top: Schematic showing the part of the hPSC colonies that was imaged using structured illumination microscopy (SIM). Bottom Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff and soft substrates, with or without the addition of soluble laminin, and stained for BMPR1A and with HCS dye staining the cytoplasm. Images were reconstructed x-z (i.e., along the apicobasal axis), and the top 10 μm of the apicobasal axis was imaged; 40 imaging planes were projected in y-axis. Scale bar = 10 μm. h. Plot of the fraction of BMPR1A-positive pixels against the distance from the cell surface. The continuous line represents the mean signal intensity and edges of the shaded areas represent +/- standard deviation from N = 3 biological replicates. i. Representative immunofluorescence images (maximum projection) of colony top (top 5 confocal planes) of hPSCs on stiff substrates, with or without soluble laminin addition into the medium, stained for Claudin-6. Scale bar = 50 μm; zoomed in images scale bar = 20 μm. j. Quantification of junctional Claudin-6 signal intensity across maximum projection images. Individual data came from N = 3 independent experiments representing biological replicates. P-value determined by Welch’s t-test. **** indicates p-value < 0.0001. k. Representative immunofluorescence images (maximum projection) of colony bottom (bottom 5 confocal planes) of hPSCs on stiff and soft substrates, in control condition or upon addition of soluble laminin into the cell medium, stained for pFAK and F-Actin. Scale bar = 50 μm. l. Quantification of normalised signal intensity in pFAK focal points in hPSCs on stiff substrates in control condition and upon addition of soluble laminin into the cell medium. Individual data came from N = 3 independent experiments representing biological replicates. P-value determined by Welch’s t-test. **** indicates p-value < 0.0001.

Article Snippet: The following primary antibodies were used: Brachyury (1:500; Cell Signalling Technology, #81694S), integrin β5 (1:200; Cell Signalling Technology, #3629S), pFAK (1:200; Thermo Scientific, #44-624G), active integrin β1 (1:100; Abcam, #ab30394), ZO-1 (1:250; Thermo Scientific, #33-9100), BMPR1A (1:50; Santa Cruz Biotechnology, #sc-518037), Myosin Regulatory Light Chain 2 (1:100; Cell Signalling Technology, #3672S), Claudin-6 (1:100; Santa Cruz Biotechnology, #sc-17669), laminin (1:200; Thermo Scientific, #PA1-16730).

Techniques: Immunofluorescence, Staining, Standard Deviation, Confocal Microscopy, Imaging, Microscopy, Control

Journal: bioRxiv

Article Title: Basement membrane mechanics drives patterned response to developmental signalling

doi: 10.64898/2026.02.13.705301

Figure Lengend Snippet: a. Normalised counts obtained from RNA-sequencing data of hPSCs on stiff and soft substrates for each laminin ( LAM ) subunit expressed. Differential expression analysis was performed using DESeq2 with the Wald test. Data was generated from N = 3 independent experiments representing biological replicates. b. Top: Schematic depicting microvilli architecture. Bottom: Representative immunofluorescence images (maximum projection) of apical surfaces (top 10 slices) of hPSCs on stiff and soft substrates, stained for pERM. Scale bar = 50 μm. c. Quantification of pERM signal intensity across maximum projection images. Individual data came from N = 3 independent experiments representing biological replicates. P-value determined by Welch’s t-test. **** indicates p-value < 0.0001. d. Representative immunofluorescence images of apical colony surface of hPSCs on stiff and soft substrates, stained for laminin and DAPI, and colony middle stained for DAPI. Scale bar = 25 μm. e. Top: Schematic representing the experimental protocol involving inhibition of integrin activity with antibodies, prior to soluble laminin addition into the cell medium and BMP4 treatment. Bottom: Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff substrates stained for pSMAD1/5 and DAPI after 1 h of BMP4 treatment, treated or untreated with soluble laminin and treated or untreated with the anti-integrin β1 and anti-integrin α6 antibodies. Scale bar = 50 μm. f. Quantification of normalised pSMAD1/5 signal intensity from immunofluorescence images of hPSCs on stiff and soft substrates after 1 h of BMP4 treatment in the control condition (blue), upon addition of soluble laminin (purple) and after inhibition of integrin β1 (pink) and both integrin β1 and integrin α6 inhibition (yellow) before soluble laminin addition. The signal intensity in each nucleus is plotted as a function of the distance of that nucleus from the colony edge. The signal intensity is normalized to the 98 th percentile. The continuous line represents the mean signal intensity and edges of the shaded areas represent +/- standard deviation from N = 3 biological replicates.

Article Snippet: The following primary antibodies were used: Brachyury (1:500; Cell Signalling Technology, #81694S), integrin β5 (1:200; Cell Signalling Technology, #3629S), pFAK (1:200; Thermo Scientific, #44-624G), active integrin β1 (1:100; Abcam, #ab30394), ZO-1 (1:250; Thermo Scientific, #33-9100), BMPR1A (1:50; Santa Cruz Biotechnology, #sc-518037), Myosin Regulatory Light Chain 2 (1:100; Cell Signalling Technology, #3672S), Claudin-6 (1:100; Santa Cruz Biotechnology, #sc-17669), laminin (1:200; Thermo Scientific, #PA1-16730).

Techniques: RNA Sequencing, Quantitative Proteomics, Generated, Immunofluorescence, Staining, Inhibition, Activity Assay, Control, Standard Deviation

Journal: Communications Biology

Article Title: Irisin regulates the phosphorylation of glucocorticoid receptor Ser212 and Ser234 and mediates glucocorticoid-induced muscle atrophy in mice

doi: 10.1038/s42003-025-09203-4

Figure Lengend Snippet: A Western blot analyses of integrin αV, integrin β5 and GAPDH in Dex and irisin-administered myotubes. B Protein levels were analyzed, n = 4 independent replicates. C Western blot analyses of P-ERK, P-GR(Ser212), P-JNK (Thr183/Tyr185), P-GR(Ser234), IGF-1, MSTN and GAPDH in integrin αVβ5 inhibitor cliengitide treatment myotube. D – I Protein levels were analyzed, n = 4 independent replicates. Data are presented as means ± SEM. One-way ANOVA was used for statistical analysis ( B ), Two-way ANOVA was used for statistical analysis ( D – I ).

Article Snippet: NC membranes were incubated with the following primary antibodies overnight at 4 °C: anti-irisin (1:1500, Phoenix Biotech, H-067-17), anti-Atrogin-1 (1:1000, abcam, ab168372), anti-MuRF-1 (1:1000, abcam, ab183094), anti-p-AKT(Ser473) (1:1000, CST, 9271S), anti-AKT (1:1000, CST, 9272S), anti-GAPDH(1:1000, ZSGBBIO, TA-08), anti-GR (1:1000, CST, 12041S), anti-Histone H3 (1:1000, CST, 3638S), anti-MSTN (1:250, abcam, ab98337), anti-p-Smad3 (1:1000, CST, 9520S), anti-Smad3 (1:1000, CST, 9523S), anti-IGF-1(1:1000, BOSTER, BA0939), anti-p-GR Ser234 (1:1000, orthologous to serine 226 in human, CST, 97285S), anti-p-GR Ser212 (1:1000, orthologous to serine 203 in human, Thermo Fisher, PA5-104446), anti-p-GR Ser134 (1:1000, orthologous to serine 143 in human, CST, 85060S), anti-p-JNK(Thr183/Tyr185) (1:1000, CST, 9251S), anti-p44/42 MAPK (Erk1/2) (1:1000, CST, 4695T), anti-integrin αV(1:1000, abcam, ab179475), anti-integrin β5(1:1000, CST, 3629S).

Techniques: Western Blot