α-smooth muscle antibody a5228  (Millipore)

Journal: Biochemical Journal

Article Title: Gα12 and Gα13 proteins are required for transforming growth factor-β-induced myofibroblast differentiation

doi: 10.1042/BCJ20240317

Figure Lengend Snippet: ( A ) Representative images and quantification of western blot analyses of myofibroblast markers in human lung fibroblasts (HLF). HLF were serum-starved for 48 h and then treated with vehicle or 1 µg/ml cholera toxin (CTX), immediately followed by treatment with vehicle or 1 ng/ml TGF-β for additional 48 h, as indicated. HFL lysates were analyzed by western blotting using antibodies recognizing collagen 1A1 (Col1A1, A2), fibronectin (FN, A3) and smooth muscle α-actin (SMA, A4). The relative immunoreactivity values were normalized to the average signal of control samples. ( B ) RT-qPCR analysis of myofibroblast markers in HLF treated with vehicle, 1 µg/ml cholera toxin (CTX), and/or 1 ng/ml TGF-β (as in A ) for 24 h. mRNA levels of COL1A1 (B1), FN1 (B2), and ACTA2 (B3) were normalized within-the-sample to the levels of housekeeping ribosomal RPL13 mRNA and compared with an average expression in control samples. ( C ) Representative images and quantification of western blot analyses of Smad2/3 phosphorylation in HLF pretreated with vehicle or 1 µg/ml CTX for 2 h followed by 30-min exposure to vehicle or 1 ng/ml TGF-β. Cell lysates were analyzed by western blotting with antibodies recognizing Smad2/3 and their phosphorylated forms as indicated. pSmad/Smad ratios were quantified for Smad2 (C2) and Smad3 (C3). ( D ) Western blot analysis of PKA-dependent phosphorylation in HFL treated as in C . and then probed with antibody recognizing phosphorylated PKA substrates (representative of three experiments). Quantitation data in A–C are the mean values ± SD of four independent treatments per group. * P < 0.05, *** P < 0.001, **** P < 0.0001, one-way ANOVA with Tukey correction for multiple comparisons.

Article Snippet: The following antibodies for Western blotting were from Millipore-Sigma: SMA (A5228, 10,000×), β-actin (A5441, 10,000×), α-tubulin (T6074, 10,000×).

Techniques: Western Blot, Control, Quantitative RT-PCR, Expressing, Phospho-proteomics, Quantitation Assay

Journal: Biochemical Journal

Article Title: Gα12 and Gα13 proteins are required for transforming growth factor-β-induced myofibroblast differentiation

doi: 10.1042/BCJ20240317

Figure Lengend Snippet: ( A ) Representative images and quantification of western blot analyses of HLF pretreated overnight with 100 ng/ml pertussis toxin (PTX), followed by treatment with either vehicle or 1 ng/ml TGF-β for 48 h. HLF lysates were analyzed using antibodies recognizing Col1A1 ( A1 ), FN ( A2 ), and SMA ( A3 ). The relative luminescence values were normalized to the average values of controls. Data are the mean values ± SD from four independent cultures per treatment. *** P < 0.001; **** P < 0.001, one-way ANOVA with Tukey correction for multiple comparisons. ( B ) Representative images of western blot analyses of the PTX pretreated HFL with or without subsequent 5-min treatment with 100 nM endothelin-1 (ET1). Cell lysates were probed with antibodies recognizing p-Erk1/2 or total Erk2. ( B1 ) Quantification of experiments presented in B . **** P < 0.001, one-way ANOVA with Tukey correction for multiple comparisons.

Article Snippet: The following antibodies for Western blotting were from Millipore-Sigma: SMA (A5228, 10,000×), β-actin (A5441, 10,000×), α-tubulin (T6074, 10,000×).

Techniques: Western Blot

Journal: Biochemical Journal

Article Title: Gα12 and Gα13 proteins are required for transforming growth factor-β-induced myofibroblast differentiation

doi: 10.1042/BCJ20240317

Figure Lengend Snippet: ( A ) Representative images and quantification of western blot analyses of HLF transfected overnight with either control siRNA (siC) or with siRNAs targeting Gαq and Gα11. HFL were next serum starved for 48 h, followed by the treatment with either vehicle or 1 ng/ml TGF-β for 48 h. Cell lysates were analyzed using antibodies recognizing Gαq/Gα11 ( A1 ), Col1A1 ( A2 ), FN ( A3 ), and SMA ( A4 ). The relative luminescence values were normalized to the average siC-treated control samples. Data are the mean values ± SD from four independent cultures per treatment. ** P < 0.01; **** P < 0.001, one-way ANOVA with Tukey correction for multiple comparisons.

Article Snippet: The following antibodies for Western blotting were from Millipore-Sigma: SMA (A5228, 10,000×), β-actin (A5441, 10,000×), α-tubulin (T6074, 10,000×).

Techniques: Western Blot, Transfection, Control

Journal: Biochemical Journal

Article Title: Gα12 and Gα13 proteins are required for transforming growth factor-β-induced myofibroblast differentiation

doi: 10.1042/BCJ20240317

Figure Lengend Snippet: ( A ) Representative images and quantification of western blot analyses of HLF transfected overnight with control siRNA (siC, 10 nM), or combination of siC (5 nM) plus siGα12 (5 nM), or siC (5 nM) plus siGα13 (5 nM), or siGα12 (5 nM) plus siGα13 (5 nM). Cells were next serum starved for 48 h, and further treated with either vehicle or 1 ng/ml TGF-β for additional 48 h. HFL lysates were analyzed by western blotting using antibodies recognizing Gα12 ( A1 ), Gα13 ( A2 ), Col1A1 ( A3 ), FN ( A4 ), SMA ( A5 ), or tubulin (A6 ). Data are the mean values ± SD from three independent cultures per treatment. * P < 0.05, ** P < 0.01; *** P < 0.001; **** P < 0.001, one-way ANOVA with Tukey correction for multiple comparisons.

Article Snippet: The following antibodies for Western blotting were from Millipore-Sigma: SMA (A5228, 10,000×), β-actin (A5441, 10,000×), α-tubulin (T6074, 10,000×).

Techniques: Western Blot, Transfection, Control

Journal: Cell Cycle

Article Title: Inhibition of endogenous hydrogen sulfide production reduces activation of hepatic stellate cells via the induction of cellular senescence

doi: 10.1080/15384101.2024.2345477

Figure Lengend Snippet: Antibodies used for Western blotting.

Article Snippet: ACTA2 , Monoclonal mouse , 1:5000 , A5228, Sigma Aldrich.

Techniques: Western Blot