antibodies against α sma acta2  (Novus Biologicals)

Journal: Gene Expression

Article Title: NOTCH4 Is a New Player in the Development of Pulmonary Fibrosis

doi: 10.14218/ge.2024.00006

Figure Lengend Snippet: Fig. 1. Induction of fibrogenesis-associated genes in human lung fibroblasts on day 3 after N4ICD transduction. contr – untransduced fibroblasts; pCIG – control vector transduced fibroblasts; N4ICD – N4ICD-transduced fibroblasts. (a) Expression efficiency of N4ICD transduction was evaluated by RT-PCR analysis with primers for N4ICD. *** p < 0.001. (b) Morphological changes in N4ICD-transduced cells compared to control ones. (c) RT-PCR results for SNAI1, SNAI2, ACTA2, and COL1A1 in control, pCIG- and N4ICD-transduced cells. *** p < 0.001; ** p < 0.01. (d) and (e). Immunofluorescence labeling analysis of control, pCIG- and N4ICD-transduced cells. Nuclei were stained with DAPI (blue). (d) Immunofluorescence staining with SNAI1 antibodies (green). (e) Im- munofluorescence staining with ACTA2 antibodies (red). DAPI, 4′,6-diamidino-2-phenylindole; mRNA, messenger RNA; N4ICD, NOTCH4 intracellular domain; RT-PCR, real-time polymerase chain reaction.

Article Snippet: Following fixation, fibroblasts were permeabilized with a 0.1% Triton X-100 solution for 10 m, washed with PBS, and blocked in 1% BSA for 1 h. Cell incubation with primary antibodies against α-SMA (ACTA2) (1:250, NB300978, Novus, USA) or SNAIL (1:250, MA5-14801, Invitrogen, USA) was performed in a humid chamber for 1 h at room temperature.

Techniques: Transduction, Control, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Labeling, Staining, Real-time Polymerase Chain Reaction

Journal: Gene Expression

Article Title: NOTCH4 Is a New Player in the Development of Pulmonary Fibrosis

doi: 10.14218/ge.2024.00006

Figure Lengend Snippet: Fig. 2. The reciprocal impact of Notch4 and TGFβ1 pathways on each other. Control – untreated fibroblasts; pCIG – control vector transduced fibroblasts; N4ICD – N4ICD-transduced fibroblasts, TGFβ1 – treated lung fibroblasts. (a) Western blot analysis of control, pCIG- and N4ICD-transduced and TGFβ1 – treat- ed fibroblast after 48 h after treatment. Membranes were incubated with Smad2, phosphorylated SMAD2 (pSmad), and b-actin antibodies. (b)-(d) Gene expression evaluation in TGFβ1-treated or N4ICD-induced lung fibroblasts on day 3 by RT-PCR. Expression in TGFβ1-treated cells is shown relative to control cells; expression in N4ICD-induced cells is shown relative to pCIG-transduced cells. *** p < 0.001; ** p < 0.01; * p < 0.05. (b) TGFβ1 and TGFBR1 expression in TGFβ1-treated or N4ICD-induced lung fibroblasts. (c) Fibrogenesis-associated genes SNAI1, SNAI2, ACTA2, and COL1A1 expression in TGFβ1-treated lung fibroblasts. (d) HEY1, NOTCH1, NOTCH2, NOTCH3, NOTCH4 expression in TGFβ1-treated or N4ICD-induced lung fibroblasts. N4ICD, Notch4 intracellular do- main; RT-PCR, real-time polymerase chain reaction; SMAD2, smad family member 2; TGFβ1, transforming growth factor beta 1.

Article Snippet: Following fixation, fibroblasts were permeabilized with a 0.1% Triton X-100 solution for 10 m, washed with PBS, and blocked in 1% BSA for 1 h. Cell incubation with primary antibodies against α-SMA (ACTA2) (1:250, NB300978, Novus, USA) or SNAIL (1:250, MA5-14801, Invitrogen, USA) was performed in a humid chamber for 1 h at room temperature.

Techniques: Control, Plasmid Preparation, Western Blot, Incubation, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in pharmacology

Article Title: Activation of BK Channels Prevents Hepatic Stellate Cell Activation and Liver Fibrosis Through the Suppression of TGFβ1/SMAD3 and JAK/STAT3 Profibrotic Signaling Pathways.

doi: 10.3389/fphar.2020.00165

Figure Lengend Snippet: FIGURE 1 | Large-conductance and Ca2+-activated K+ (BK) channels are expressed and functional in activated hepatic stellate cells (HSCs). (A,B) Representative RT-qPCR (A) and western blots (B) showing the expression of ACTA2 and the BK channels alpha subunit KCNMA1 in activated human LX2 cells treated with transforming growth factor beta 1 (TGFβ1) and spontaneously activated primary rat HSCs in vitro. (C) Representative immunofluorescence images of KCNMA1 and ACTA2 in activated primary rat HSCs (Scale bars, 25 µm). (D,E) Representative whole-cell K+ current traces and the normalized current recorded from activated HSCs before and after treatment with rottlerin (Rot) and paxilline (Pax), as indicated at 1 µM internal Ca2+. The whole-cell currents were elicited by 1 s voltage ramps from −100 to 80 mV. Rottlerin (1 µM) and paxilline (10 µM) were freshly prepared from stock solutions and the final DMSO concentration was 0.1% (*p < 0.05 compared with the vehicle group, n = 3).

Article Snippet: Antibodies against SMAD3, p-SMAD3, JAK2, p-JAK2, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, United States), the anti-α-SMA (ACTA2) antibody was from Novus Biology (Centennial, CO, United States), and the anti-BK-Slo1 was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Functional Assay, Quantitative RT-PCR, Western Blot, Expressing, In Vitro, Concentration Assay

Journal: Frontiers in pharmacology

Article Title: Activation of BK Channels Prevents Hepatic Stellate Cell Activation and Liver Fibrosis Through the Suppression of TGFβ1/SMAD3 and JAK/STAT3 Profibrotic Signaling Pathways.

doi: 10.3389/fphar.2020.00165

Figure Lengend Snippet: FIGURE 2 | Overexpression of the BK channel alpha subunit (KCNMA1) inhibits hepatic stellate cell (HSC) migration and fibrosis-related gene expression. (A) Validation of KCNMA1 overexpression by qPCR (left) and western blotting (right). (B) Representative images of cells (left) and average number of migrated cells (right) showing the inhibitory effects of transient KCNMA1 overexpression on LX2 cell migration. (C) Normalized mRNA expression of aortic smooth muscle actin (ACTA2), collagen type I alpha 1 chain (COL1A1), COL1A2, and C-C motif chemokine ligand 2 (CCL2) in LX2 cells pretreated with TGFβ1 after transfection with KCNMA1-expressing plasmids. (D) Detection of KCNMA1 by qPCR (left) and western blotting (right) after siRNA transfection. (E) Representative images (left) and averaged number of migrated cells (right) showing the inhibitory effects of knocking down KCNMA1 on LX2 cell migration. (F) Normalized mRNA expression levels of ACTA2, COL1A1, COL1A2, and CCL2 in LX2 cells pretreated with TGFβ1 after transfection with siRNA targeting KCNMA1. (G) Ratio of EdU-positive cells to Hoechst 33342-stained nuclei after transfection of KCNMA1-expressing plasmids (left) or KCNMA1 siRNA (right) (∗p < 0.05, ∗∗p < 0.01 compared with the vector or negative control (NC) group.

Article Snippet: Antibodies against SMAD3, p-SMAD3, JAK2, p-JAK2, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, United States), the anti-α-SMA (ACTA2) antibody was from Novus Biology (Centennial, CO, United States), and the anti-BK-Slo1 was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Over Expression, Migration, Gene Expression, Biomarker Discovery, Western Blot, Expressing, Transfection, Staining, Plasmid Preparation, Negative Control

Journal: Frontiers in pharmacology

Article Title: Activation of BK Channels Prevents Hepatic Stellate Cell Activation and Liver Fibrosis Through the Suppression of TGFβ1/SMAD3 and JAK/STAT3 Profibrotic Signaling Pathways.

doi: 10.3389/fphar.2020.00165

Figure Lengend Snippet: FIGURE 3 | Upregulation of large-conductance and Ca2+-activated K+ (BK) channel activity by rottlerin inhibits hepatic stellate cell (HSC) migration and fibrosis-related gene expression in vitro. (A,B) Representative images (left) and averaged number of migrated cells (right) showing the inhibitory effects of rottlerin (Rot), a BK channel activator, on the migration of human LX2 cells (A) and activated primary rat HSCs (B) at the indicated concentrations (∗p < 0.05, ∗∗p < 0.01 compared with vehicle). (C) Relative mRNA expression levels of ACTA2, COL1A1, COL1A2, and CCL2 in human LX2 cells with the indicated treatments for 24 h. (D) Normalized cell viability of LX2 cells measured under the indicated conditions (∗∗p < 0.01 compared with vehicle, ##p < 0.01 compared with the TGFβ1-treated group).

Article Snippet: Antibodies against SMAD3, p-SMAD3, JAK2, p-JAK2, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, United States), the anti-α-SMA (ACTA2) antibody was from Novus Biology (Centennial, CO, United States), and the anti-BK-Slo1 was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Activity Assay, Migration, Gene Expression, In Vitro, Expressing

Journal: Frontiers in pharmacology

Article Title: Activation of BK Channels Prevents Hepatic Stellate Cell Activation and Liver Fibrosis Through the Suppression of TGFβ1/SMAD3 and JAK/STAT3 Profibrotic Signaling Pathways.

doi: 10.3389/fphar.2020.00165

Figure Lengend Snippet: FIGURE 4 | Treatment with the large-conductance and Ca2+-activated K+ (BK) channel activator rottlerin ameliorates CCl4-induced liver fibrosis in vivo. (A) Serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) from rats in the indicated groups (∗∗p < 0.01 compared with the vehicle (Veh) group, #p < 0.05 and ##p < 0.01 compared with the CCl4-treated group, n = 5). (B) Relative mRNA levels of ACTA2 in the liver tissue of rats subjected to the indicated treatments (∗∗p < 0.01 compared with the Veh group, ##p < 0.01 compared with the CCl4-treated group, n = 5). (C) Representative western blots (left) and normalized intensity (right) of the indicated ACTA2 protein levels in rats subjected to the indicated treatments (∗∗p < 0.01 compared with the Veh group, ##p < 0.01 compared with the CCl4-treated group, n = 5). (D) Representative images of liver sections stained with hematoxylin and eosin (H&E), anti-ACTA2 antibody, and Sirius Red (left) and quantification of the positive area (right), in rats subjected to the indicated treatments (∗∗p < 0.01 compared with the Veh group, ##p < 0.01 compared with the CCl4-treated group, scale bar: 50 µm, n = 5).

Article Snippet: Antibodies against SMAD3, p-SMAD3, JAK2, p-JAK2, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, United States), the anti-α-SMA (ACTA2) antibody was from Novus Biology (Centennial, CO, United States), and the anti-BK-Slo1 was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: In Vivo, Western Blot, Staining

Journal: Tissue Engineering. Part A

Article Title: Comparison of Different In Vivo Incubation Sites to Produce Tissue-Engineered Small Intestine

doi: 10.1089/ten.tea.2017.0313

Figure Lengend Snippet: Sources and Dilutions of Primary and Secondary Antibodies Used for Immunofluorescence Staining

Article Snippet: GFP, green fluorescent protein; IF, immunofluorescence; PGA, polyglycolic acid; PLLA, poly-L-lactic acid; TESI, tissue-engineered small intestine. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Primary antibodies Description Species raised Source Catalog no. Dilution α-Smooth muscle actin Mouse Novus Biologicals NBP2-44463 1:100 CD31 Rabbit Abcam Ab182981 1:400 Chromogranin A Rabbit Abcam ab15160 1:200 E-cadherin Mouse Abcam ab76055 1:60 E-cadherin Goat LifeSpan Biosciences LS- B12414 1:200 GFAP Goat Abcam ab53554 1:100 GFP Chicken Novus Biologicals NB100-1614 1:100 GFP Rabbit Novus Biologicals NB600-308 1:200 Ki67 Rabbit Novus Biologicals NB110-89717 1:100 Lysozyme Rabbit LifeSpan Biosciences LS-C407874 1:200 TUJ1 Mouse Promega G712a 1:600 Villin Rabbit LifeSpan Biosciences LS-C407669 1:200 Open in a separate window Secondary antibodies Description Species raised Source Catalog no. Dilution Alexa Fluor ® 488 AffiniPure anti-chicken IgG Donkey Jackson ImmunoResearch 703-545-155 1:100 Alexa Fluor ® 488 AffiniPure anti-goat IgG Donkey Jackson ImmunoResearch 705-545-147 1:100 CyTM3 AffiniPure Anti-goat IgG Donkey Jackson ImmunoResearch 705-165-147 1:100 CyTM3 AffiniPure Anti-mouse IgG Donkey Jackson ImmunoResearch 715-165-151 1:100 Alexa Fluor ® 647 AffiniPure anti-mouse IgG Donkey Jackson ImmunoResearch 715-605-151 1:100 Alexa Fluor ® 488 AffiniPure anti-rabbit IgG Donkey Jackson ImmunoResearch 711-545-152 1:100 CyTM3 AffiniPure anti-rabbit IgG Donkey Jackson ImmunoResearch 711-165-152 1:100 Open in a separate window GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein.

Techniques: Immunofluorescence

Journal: Tissue Engineering. Part A

Article Title: Comparison of Different In Vivo Incubation Sites to Produce Tissue-Engineered Small Intestine

doi: 10.1089/ten.tea.2017.0313

Figure Lengend Snippet: TESI IF to detect blood vessel density and cell source. IF staining of TESI produced in the following implantation sites: (A) omentum; (B) mesentery; (C) uterine horn membrane; (D) abdominal wall; and (E) subcutaneous space. Sections were stained for CD31 (red) to detect endothelial cells, α-smooth muscle actin (α-SMA; green) to detect SMC, and E-cadherin (ECAD; gray) to detect intestinal epithelial cells. GFP staining was used to track the source of the cells in blood vessels. Blood vessels were identified by α-SMA and CD31 staining (F and G respectively), and then the source of the cells identified by GFP staining (H). Blood vessels originating from implanted cells were α-SMA (+) CD31 (+) GFP (+) whereas blood vessels originating from the host were α-SMA (+) CD31 (+) GFP (−). (I) DAPI staining to detect cell nuclei; (J) merged image. Scale bar = 50 μm. d, blood vessel derived from donor cells; h, blood vessel derived from host cells. SMA, smooth muscle actin; SMC, smooth muscle cell.

Article Snippet: GFP, green fluorescent protein; IF, immunofluorescence; PGA, polyglycolic acid; PLLA, poly-L-lactic acid; TESI, tissue-engineered small intestine. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Primary antibodies Description Species raised Source Catalog no. Dilution α-Smooth muscle actin Mouse Novus Biologicals NBP2-44463 1:100 CD31 Rabbit Abcam Ab182981 1:400 Chromogranin A Rabbit Abcam ab15160 1:200 E-cadherin Mouse Abcam ab76055 1:60 E-cadherin Goat LifeSpan Biosciences LS- B12414 1:200 GFAP Goat Abcam ab53554 1:100 GFP Chicken Novus Biologicals NB100-1614 1:100 GFP Rabbit Novus Biologicals NB600-308 1:200 Ki67 Rabbit Novus Biologicals NB110-89717 1:100 Lysozyme Rabbit LifeSpan Biosciences LS-C407874 1:200 TUJ1 Mouse Promega G712a 1:600 Villin Rabbit LifeSpan Biosciences LS-C407669 1:200 Open in a separate window Secondary antibodies Description Species raised Source Catalog no. Dilution Alexa Fluor ® 488 AffiniPure anti-chicken IgG Donkey Jackson ImmunoResearch 703-545-155 1:100 Alexa Fluor ® 488 AffiniPure anti-goat IgG Donkey Jackson ImmunoResearch 705-545-147 1:100 CyTM3 AffiniPure Anti-goat IgG Donkey Jackson ImmunoResearch 705-165-147 1:100 CyTM3 AffiniPure Anti-mouse IgG Donkey Jackson ImmunoResearch 715-165-151 1:100 Alexa Fluor ® 647 AffiniPure anti-mouse IgG Donkey Jackson ImmunoResearch 715-605-151 1:100 Alexa Fluor ® 488 AffiniPure anti-rabbit IgG Donkey Jackson ImmunoResearch 711-545-152 1:100 CyTM3 AffiniPure anti-rabbit IgG Donkey Jackson ImmunoResearch 711-165-152 1:100 Open in a separate window GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein.

Techniques: Staining, Produced, Membrane, Derivative Assay

Journal: Tissue Engineering. Part A

Article Title: Comparison of Different In Vivo Incubation Sites to Produce Tissue-Engineered Small Intestine

doi: 10.1089/ten.tea.2017.0313

Figure Lengend Snippet: Sources and Dilutions of Primary and Secondary Antibodies Used for Immunofluorescence Staining

Article Snippet: α-Smooth muscle actin , Mouse , Novus Biologicals , NBP2-44463 , 1:100.

Techniques: Immunofluorescence

Journal: Tissue Engineering. Part A

Article Title: Comparison of Different In Vivo Incubation Sites to Produce Tissue-Engineered Small Intestine

doi: 10.1089/ten.tea.2017.0313

Figure Lengend Snippet: TESI IF to detect blood vessel density and cell source. IF staining of TESI produced in the following implantation sites: (A) omentum; (B) mesentery; (C) uterine horn membrane; (D) abdominal wall; and (E) subcutaneous space. Sections were stained for CD31 (red) to detect endothelial cells, α-smooth muscle actin (α-SMA; green) to detect SMC, and E-cadherin (ECAD; gray) to detect intestinal epithelial cells. GFP staining was used to track the source of the cells in blood vessels. Blood vessels were identified by α-SMA and CD31 staining (F and G respectively), and then the source of the cells identified by GFP staining (H). Blood vessels originating from implanted cells were α-SMA (+) CD31 (+) GFP (+) whereas blood vessels originating from the host were α-SMA (+) CD31 (+) GFP (−). (I) DAPI staining to detect cell nuclei; (J) merged image. Scale bar = 50 μm. d, blood vessel derived from donor cells; h, blood vessel derived from host cells. SMA, smooth muscle actin; SMC, smooth muscle cell.

Article Snippet: α-Smooth muscle actin , Mouse , Novus Biologicals , NBP2-44463 , 1:100.

Techniques: Staining, Produced, Membrane, Derivative Assay