anti collagen 1α2  (Novus Biologicals)

Journal: JCI Insight

Article Title: The CLCA1/TMEM16A/Cl – current axis associates with H 2 S deficiency in diabetic kidney injury

doi: 10.1172/jci.insight.174848

Figure Lengend Snippet: ( A ) Renal cortical expression of CLCA1 and TMEM16A was increased in diabetic mice compared with nondiabetic controls, and it was inhibited by the administration of NaHS for 3 weeks. ( B ) Renal cortical H 2 S content was decreased in diabetic mice. ( C ) H 2 S generation was decreased in the kidney cortex of db/db diabetic mice by 45% compared with db/m control mice (0.10 ± 0.01 vs. 0.18 ± 0.01 nmol/g/min, mean ± SD, respectively, P < 0.001). ( D ) Blood glucose levels were not affected by NaHS. ( E ) Diabetes-induced increase in renal cortical content of collagen 1α2 and fibronectin was inhibited by NaHS. ( F ) Phosphorylation of p70 S6 kinase (P-S6K) was increased in the renal cortex of diabetic mice, indicating mTORC1 activation; it was inhibited by NaHS. ( G ) Diabetes-induced increase in urinary albumin-to-creatinine ratio (urinary ACR) was reduced by NaHS. Data (mean ± SD) from 9 mice in each group of mice (db/m nondiabetic controls treated with or without NaHS, db/db diabetic mice treated with or without NaHS) are shown in bars with scatterplots and were analyzed ( A and D – F ) by ANOVA. Data from 3 mice in each group are presented ( B and C ) and were analyzed by t test. * P < 0.05, ** P < 0.01, *** P < 0.001. ( H ) Renal tubular TMEM16A expression significantly correlated with the degree of albuminuria in 49 American Indian individuals with diabetes ( P = 0.0059 by ANOVA). NormALB, normo-albuminuria; microALB, micro-albuminuria; and macroALB, macroalbuminuria. ( I ) There was a significant correlation between TMEM16A mRNA content in the kidney tubulointerstitium and urinary albumin-to-creatinine ratio by Pearson’s correlation coefficient. ( J ) TMEM16A expression in the human kidneys of control ( n = 3) and diabetes individuals ( n = 4).

Article Snippet: We employed antibodies against the following: CLCA1 (catalog, ab180851, Abcam), TMEM16A (catalog, ab72984, Abcam), fibronectin (catalog, ab2413, Abcam), collagen 1α2 (catalog, 14695-1-AP, Proteintech Group), laminin-γ1 (catalog, sc-5584, Santa Cruz Biotechnology), phospho-Thr389-p70 S6 kinase (catalog, 9205, Cell Signaling Technology), p70 S6 kinase (catalog, 9202, Cell Signaling Technology), IL-6 (catalog, ab7737, Abcam), phospho-Tyr705-STAT3 (catalog, ab76315, Abcam), STAT3 (catalog, ab68153, Abcam), and actin (catalog, A2066, MilliporeSigma).

Techniques: Expressing, Control, Phospho-proteomics, Activation Assay

Journal: JCI Insight

Article Title: The CLCA1/TMEM16A/Cl – current axis associates with H 2 S deficiency in diabetic kidney injury

doi: 10.1172/jci.insight.174848

Figure Lengend Snippet: ( A and B ) High glucose–induced increase in fibronectin and collagen 1α2 was inhibited by reduced expression of CLCA1. ( C and D ) siRNA-mediated reduced CLCA1 expression did not permit mTORC1 activation by high glucose; however, it did not affect high glucose inhibition of AMPK activity. ACC, acetyl-CoA carboxylase. Data from 3–4 experiments (mean ± SD) are shown in bars with scatterplots and were analyzed by ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: We employed antibodies against the following: CLCA1 (catalog, ab180851, Abcam), TMEM16A (catalog, ab72984, Abcam), fibronectin (catalog, ab2413, Abcam), collagen 1α2 (catalog, 14695-1-AP, Proteintech Group), laminin-γ1 (catalog, sc-5584, Santa Cruz Biotechnology), phospho-Thr389-p70 S6 kinase (catalog, 9205, Cell Signaling Technology), p70 S6 kinase (catalog, 9202, Cell Signaling Technology), IL-6 (catalog, ab7737, Abcam), phospho-Tyr705-STAT3 (catalog, ab76315, Abcam), STAT3 (catalog, ab68153, Abcam), and actin (catalog, A2066, MilliporeSigma).

Techniques: Expressing, Activation Assay, Inhibition, Activity Assay

Journal: JCI Insight

Article Title: The CLCA1/TMEM16A/Cl – current axis associates with H 2 S deficiency in diabetic kidney injury

doi: 10.1172/jci.insight.174848

Figure Lengend Snippet: ( A and B ) siRNA against TMEM16A abolished high glucose–induced increased expression of fibronectin and collagen 1α2. ( C and D ) TMEM16A inhibitor, T16Ainh-A01, also suppressed high glucose–stimulated expression of fibronectin and collagen 1α2. ( E and F ) TMEM16A siRNA partly abolished mTORC1 activation by high glucose but not high glucose inhibition of AMPK activity. Data from 3–4 experiments (mean ± SD) are shown in bars with scatterplots and were analyzed by ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: We employed antibodies against the following: CLCA1 (catalog, ab180851, Abcam), TMEM16A (catalog, ab72984, Abcam), fibronectin (catalog, ab2413, Abcam), collagen 1α2 (catalog, 14695-1-AP, Proteintech Group), laminin-γ1 (catalog, sc-5584, Santa Cruz Biotechnology), phospho-Thr389-p70 S6 kinase (catalog, 9205, Cell Signaling Technology), p70 S6 kinase (catalog, 9202, Cell Signaling Technology), IL-6 (catalog, ab7737, Abcam), phospho-Tyr705-STAT3 (catalog, ab76315, Abcam), STAT3 (catalog, ab68153, Abcam), and actin (catalog, A2066, MilliporeSigma).

Techniques: Expressing, Activation Assay, Inhibition, Activity Assay

Journal: Aging Cell

Article Title: Tendinosis develops from age‐ and oxygen tension‐dependent modulation of Rac1 activity

doi: 10.1111/acel.12934

Figure Lengend Snippet: Aged and diseased tendon display altered cellular morphology and extracellular matrix production in vivo. (a–c) Intra‐operative photographs of (a) 84‐year‐old female patient undergoing fibrocartilage pulley release for recalcitrant flexor tendinosis (aged), (b) 42‐year‐old female patient undergoing tendinosis excision prior to reconstruction (tendinosis), (c) 30‐year‐old female patient undergoing primary tendon repair after acute laceration (young). Forcep tip delineates the flexor tendon. (d–f) Alcian blue (AB) histopathologic staining of (a‐c), respectively. Bar = 20 μm. (g–i) Alizarin red (AR) histopathologic staining of (a‐c), respectively. Bar = 20 μm. (j–u) Immunohistochemical staining of aged (j,m,p,s), tendinosis (k,n,q,t), and young (l,o,r,u) patient samples as in (a–c) for: col1α2 (j‐l), col2α1 (m–o), colX (p–r) and HIF‐1α (s–u). Bar = 20 μm. (v) Quantitation of immunoblot of collagen 1α2 and collagen 2α1 expression from young asymptomatic tendon (Young) or aged diseased tendon (Aged). * p < 0.05, n = 4. (w, x) FTIR of young asymptomatic tendon (Young) or aged diseased tendon (Aged tendinotic). (w) Protein:collagen ratio. (x) Mineral:collagen ratio. Spectrophotometric absorbance as shown below quantitation with absorbance scale to the right. * p < 0.05, n = 4

Article Snippet: For immunofluorescence, slides were incubated with primary antibodies: mouse monoclonal collagen 1α2, collagen 2α1, or collagen 3 (Abcam) at 4°C overnight.

Techniques: In Vivo, Staining, Immunohistochemical staining, Quantitation Assay, Western Blot, Expressing

Journal: Aging Cell

Article Title: Tendinosis develops from age‐ and oxygen tension‐dependent modulation of Rac1 activity

doi: 10.1111/acel.12934

Figure Lengend Snippet: Aged tenocytes display altered cellular morphology and extracellular matrix production in vitro. Human tenocytes from aged (Aged), young tendinotic (Tendinotic), and young asymptomatic (Young) patients were cultured at low (1k/cm 2 aged a‐b; 5k/cm 2 young tendinotic c, d and young e, f) cell densities in hypoxic (1% O 2 ; a, c, e) or normoxic conditions (21% O 2 ; b, d, f) over twelve weeks. Shown are phase contrast micrographs of representative cells from week 2 (wk2), week 8 (wk8), and week 12 (wk12). Bar = 50 μm. (g–j) Brightfield photomicrographs of alizarin red stained aged (g, h) or young asymptomatic (i, j) tenocytes in hypoxic (1% O 2 , g, i) or normoxic (21% O 2 , h, j) conditions at week 12. (k) Spectrophotometric quantitation of calcification (mg/dL) as detected by alizarin red staining of aged (Aged) or young asymptomatic (Young) tenocytes cultured in hypoxic (Hypoxia, 1% O 2 ) or normoxic (Normoxia, 21% O 2 ) conditions at low (Low, 1k/cm 2 for aged, 5k/cm 2 for young asymptomatic) or high (High, 25k/cm 2 for aged, 50k/cm 2 for young asymptomatic) cell densities and harvested at week 12 in culture. * p < 0.05, n = 4. (l–q) qRT–PCR expression of aged (red), young tendinotic (green), or young asymptomatic (blue) tenocyte (C1, Scl, Tm, C3), fibrochondrocyte (C2), and mineralized fibrochondrocyte (CX) markers over a 12‐week timecourse, hypoxic (1% O 2 ) as compared to normoxic (21% O 2 ) conditions, at cell densities: 1k = 1 × 10 3 cells/cm 2 ; 5k = 5 × 10 3 cells/cm 2 ; 25k = 25 × 10 3 cells/cm 2 ; 50k = 50 × 10 3 cells/cm 2 (not available for aged tenocytes). C1 = col1α2; C2 = col2α1; C3 = col3; CX = colX; Scl = scleraxis; Tm = tenomodulin

Article Snippet: For immunofluorescence, slides were incubated with primary antibodies: mouse monoclonal collagen 1α2, collagen 2α1, or collagen 3 (Abcam) at 4°C overnight.

Techniques: In Vitro, Cell Culture, Staining, Quantitation Assay, Quantitative RT-PCR, Expressing

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Autophagy and the unfolded protein response promote profibrotic effects of TGF-β 1 in human lung fibroblasts

doi: 10.1152/ajplung.00372.2017

Figure Lengend Snippet: Human airway and lung fibroblasts isolated from nonidiopathic pulmonary fibrosis (non-IPF) lungs do not differ in transforming growth factor-β1 (TGF-β1)-induced fibrotic response, unfolded protein response (UPR), and autophagy activity. A: primary human non-IPF airway and lung fibroblasts (passages 3–6) were treated with TGF-β1 (2.5 ng/ml) (0–96 h). Cell lysates were collected and the abundance of UPR (BIP), autophagy (LC3βII), and profibrotic proteins (collagen 1α2 and fibronectin) were assessed by Western blot. Protein loading was confirmed using GAPDH. A–E are representative of experiments performed on 3 different primary non-IPF airway and lung fibroblast cultures (n = 3). No differences (P > 0.05) were detected in UPR (B), fibrotic (C and D), and autophagy (E) markers between airway and lung fibroblasts. B–E: dot plots of data from individual experiments. The horizontal line in each column represents the mean, with error bars showing SE. NS, no significant difference.

Article Snippet: Goat anti-human collagen 1α2 (sc-8786, 1:1,500), rabbit anti-human/mouse/rat fibronectin (sc-9068, 1:1,000), and mouse anti-human GAPDH (sc-69778, 1:7,000) were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA).

Techniques: Isolation, Activity Assay, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Autophagy and the unfolded protein response promote profibrotic effects of TGF-β 1 in human lung fibroblasts

doi: 10.1152/ajplung.00372.2017

Figure Lengend Snippet: Fibroblasts isolated from non-IPF lungs and those with IPF donors differ in TGF-β1-induced fibrotic response, UPR, and autophagy activity. A: primary human non-IPF fibroblast and IPF fibroblasts (passages 3–6) were treated with TGF-β1 (2.5 ng/ml) (0–120 h). Cell lysates were collected and subjected to SDS-PAGE, and the expression of UPR [BIP, IRE1α, and sliced XBP1 (sXBP1)], autophagy (LC3βII and Atg5-12), and extracellular matrix (ECM) proteins (collagen 1α2 and fibronectin), as well as Smad signaling effectors were assessed by Western blot analysis. Protein loading was confirmed using GAPDH. Data in A represent experiments performed on 4 different primary non-IPF and IPF fibroblasts. B and C: densitometry showed that TGF-β1 stimulation led to an increase in abundance of collagen Iα2 and fibronectin that was greater in IPF fibroblasts compared with non-IPF fibroblasts. D and E: abundance of autophagy proteins LC3βII/LC3βI and Atg5-12 following TGF-β1 stimulation was significantly lower in IPF fibroblasts as compared with non-IPF fibroblasts. F–H: TGF-β1 stimulation also resulted in significant accumulation of UPR proteins (BIP, IRE1α, and sXBP), which was greater in IPF fibroblasts compared with non-IPF fibroblasts. Data in B–H are shown in dot plots of from 4 individual experiments using different IPF and non-IPF samples. The horizontal line in each column represents the mean ± SE. ***P < 0.001; **P < 0.01: *P < 0.05; NSP > 0.05, no significant difference. I: primary human IPF fibroblasts (passages 2–3) were treated with TGF-β1 (2.5 ng/ml) for 96 h and then treated with bafiloECM A1 (Baf-A1; 100 nM) for up to 4 h more. LC3βII accumulation and p62 degradation were measured using GAPDH as protein loading confirmed.

Article Snippet: Goat anti-human collagen 1α2 (sc-8786, 1:1,500), rabbit anti-human/mouse/rat fibronectin (sc-9068, 1:1,000), and mouse anti-human GAPDH (sc-69778, 1:7,000) were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA).

Techniques: Isolation, Activity Assay, SDS Page, Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Autophagy and the unfolded protein response promote profibrotic effects of TGF-β 1 in human lung fibroblasts

doi: 10.1152/ajplung.00372.2017

Figure Lengend Snippet: Autophagy is required for TGF-β1-induced fibrosis in both non-IPF and IPF fibroblasts. A: primary human non-IPF fibroblasts were treated with TGF-β1 (2.5 ng/ml) in the presence or absence of the chemical inhibitors of autophagy (Baf-A1; 10 nM) for the indicated times. A representative blot from 4 different primary non-IPF fibroblasts (n = 4) is shown. B and C: densitometry analysis of different IPF and non-IPF fibroblasts confirmed that the inhibition of autophagy significantly inhibited TGF-β1-induced collagen biosynthesis in human non-IPF fibroblasts while there is not any significant difference in LC3βII between TGF-β1 and TGF-β1 + Baf-A1 group. Densitometry data are normalized to time matched controls grown in insulin/transferrin/selenium medium. Data are shown in dot plots of from 4 individual experiments using different cell lines. The horizontal line in each column represents the mean, with error bars showing SE. ***P < 0.001; NSP > 0.05, no significant difference. D: primary human IPF fibroblasts were treated with TGF-β1 (2.5 ng/ml) in the presence or absence of the autophagy chemical inhibitor bafiloECM A1 (10 nM) for the indicated time points. Immunoblots show the expression of collagen 1α2 and LC3βII. Data in D represent examples of 4 different primary IPF fibroblasts (n = 4). E and F: densitometry analysis confirmed that autophagy inhibition significantly inhibited TGF-β1-induced collagen biosynthesis in human IPF fibroblasts while there is not any significant difference in LC3βII between TGF-β1 and TGF-β1 + Baf-A1 group. Densitometry data are normalized to time-matched controls in ITS medium. Data are shown in dot plots of from 4 individual experiments using different cell lines. The horizontal line in each column represents the mean, with error bars showing SE. *** P < 0.001; NSP > 0.05. no significant difference.

Article Snippet: Goat anti-human collagen 1α2 (sc-8786, 1:1,500), rabbit anti-human/mouse/rat fibronectin (sc-9068, 1:1,000), and mouse anti-human GAPDH (sc-69778, 1:7,000) were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA).

Techniques: Inhibition, Western Blot, Expressing

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Autophagy and the unfolded protein response promote profibrotic effects of TGF-β 1 in human lung fibroblasts

doi: 10.1152/ajplung.00372.2017

Figure Lengend Snippet: XBP splicing is required for TGF-β1-induced fibrosis in primary human IPF, but not non-IPF, fibroblasts. A: primary human non-IPF fibroblasts were stimulated with TGF-β1 (2.5 ng/ml) in the presence or absence of the IRE1α inhibitor MKC8866 (10 μM) for the indicated time points. The representative immunoblot shows the abundance of collagen 1α2, fibronectin, and sXBP in 4 different primary non-IPF fibroblasts. B and C: densitometry analysis (n = 4) confirmed that MKC8866 did not significantly reduce TGF-β1-induced collagen 1α2 or fibronectin biosynthesis in non-IPF human fibroblasts. Data are shown in dot plots of from 4 individual experiments using different cell lines. The horizontal line in each column represents the mean, with error bars showing SE. NSP > 0.05, no significant difference. D: primary human IPF fibroblasts were stimulated with TGF-β1 (2.5 ng/ml) in the presence or absence of IRE1 inhibitor (10 μM) for the indicated time points. Immunoblots are arranged to show expression of collagen 1α2, fibronectin and sXBP. The immunoblot is representative of experiments performed on 4 different primary IPF fibroblasts (n = 4). E and F: densitometry analysis confirmed that treatment of IPF fibroblasts with IRE1 inhibitor significantly reduced TGF-β1-induced collagen 1α2 and fibronectin biosynthesis. Data are shown in dot plots of from 4 individual experiments using different cell lines. The horizontal line in each column represents the mean, with error bars showing SE. ***P < 0.001; **P < 0.01. G: primary human IPF fibroblasts were stimulated with TGF-β1 (2.5 ng/ml) in the presence or absence of IRE1 inhibitor (10 μM) for 96 h. For a baseline control, cells were maintained in serum- and TGF-β1-deficient media. Quantitative PCR showed that MKC8866 significantly inhibited TGF-β1-induced fibronectin mRNA accumulation in IPF fibroblasts. The experiments were performed in duplicate on 2 different primary IPF fibroblast cell lines. Data are shown in dot plots from each experiment. The horizontal line in each column represents the mean, with error bars showing SE. ***P < 0.001.

Article Snippet: Goat anti-human collagen 1α2 (sc-8786, 1:1,500), rabbit anti-human/mouse/rat fibronectin (sc-9068, 1:1,000), and mouse anti-human GAPDH (sc-69778, 1:7,000) were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA).

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