Anti α sma 14395 1 ap

Formononetin (For, A0232), isorhamnetin (Iso, A0514), kaempferol (Kae, A0129), calycosin (Cal, A0190), and quercetin (Que, A0083) (purity ≥ 98%) were provided by Chengdu Must Biotechnology Co., Ltd. (Chengdu, China), which were mixed with dimethyl sulfoxide (DMSO, 0.1%, v/v; Sigma, St. Louis, MO, United States). In the meantime, the HEK293-derived recombinant human TGF-β1 (100-21) was purchased from PeproTech (New Jersey, United States). The anti-α-SMA (14395-1-AP) and anti-GAPDH (6004-1-lg) antibodies, together with the HRP-labeled AffiniPure goat anti-mouse IgG antibody (H + L) (SA00001-1), were provided by Proteintech (Wuhan, China). The HRP-labeled goat anti-rabbit IgG antibody (H + L) (BS13278) was provided by Bioworld Technology (St. Paul, MN, United States). The Alexa fluor 568-conjugated goat anti-rabbit IgG (H + L) antibody was purchased from Abcam (Cambridge, MA, United States). The enhanced BCA protein assay kit (P0010), RIPA lysis buffer (P0013) and DAPI staining solution (C1005) were provided by Beyotime Institute of Biotechnology (Shanghai, China).

Cells were transfected and/or treated and then lysed in lysis buffer (Beyotime, Shanghai, China). For nucleus protein isolation, cells were lysed by Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China) according to the manufacture's instruction. The cell protein extracts (30 ~ 50 µg) were separated by sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) electrophoretically transferred onto polyvinylidene difluoride membranes, which were then blocked with 5% non‐fat dried milk in TBS‐T and then incubated with proper primary antibodies listed below: anti‐α‐SMA (14395‐1‐AP; ProteinTech, Rosemont, IL, USA), anti‐vimentin (60330‐1‐lg; ProteinTech), anti‐E‐cadherin (20874‐1‐AP; ProteinTech), anti‐Notch1 (ab83232; Abcam), anti‐C‐MYC (ab32072; Abcam), anti‐HES1 (ab71559; Abcam), anti‐HEY1 (ab22614; Abcam), anti‐GAPDH (60004‐1‐lg, ProteinTech, USA), anti‐Histone H3(17168‐1‐AP, ProteinTech) and anti‐β‐actin (60008‐1‐lg, ProteinTech). After washing with TBST buffer, the membranes were incubated with the appropriate secondary antibodies. Immunoreactive proteins were visualized using enhanced chemiluminescence (ECL) techniques.

Kidney tissues and cell culture samples were homogenized in RIPA lysis buffer. The protein contents were measured by using a bicinchoninic acid assay. A total of 30~50 μg of protein was resolved by SDS-PAGE, transferred to PVDF membranes, and probed with specific antibodies. β-Actin was used as an internal control. The following primary antibodies were used: anti-ACSS2 (ab133664), anti-p62 (ab56416), anti-S6K1 (ab32529), and anti–p-S6K1 (ab59208) purchased from Abcam; anti-nephrin (sc-376522) purchased from Santa Cruz Biotechnology; anti–α-SMA (14395-1-AP) and anti-LC3 (14600-1-AP) purchased from Proteintech; and anti-Raptor (no. 2280), anti-mTOR (no. 2983), anti–p-mTOR (no. 5536), anti-4EBP (no. 9644), anti–p-4EBP (no. 2855), and anti-H3K9ac (no. 9649) purchased from Cell Signaling Technology. The densitometric analysis was performed using ImageJ.

Steps were performed as described in the manufacturer’s description (sp-9001, ZSGB-BIO, Beijing, China). Paraffin-embedded sections (5 μm) were deparaffinized and hydrated, and then antigen retrieval was performed in a microwave using citric acid buffer (pH 6.0). Endogenous peroxidase activity and nonspecific antigens were blocked with peroxidase-blocking reagent and goat serum, followed by incubation with primary antibodies, including anti-SCAP (ab153933, Abcam, Cambridge, UK), anti-α-SMA (14395-1-AP, Proteintech, Wuhan, China), anti-PCNA (#13110, Cell Signaling Technology, Boston, USA), and anti-CASPASE3 (#9662, Cell Signaling Technology, Boston, USA). Then, the tissues were incubated with a goat anti-rabbit IgG/TRITC secondary antibody (ZF-0316, ZSGB-BIO, Beijing, China) and subsequently incubated with streptavidin-conjugated horseradish peroxidase. The peroxidase reaction was performed using DAB substrate (ZLI-9018, ZSGB-BIO, Beijing, China) with hematoxylin nuclear counterstaining. Slices were visualized using a Pannoramic Flash DESK DX (3DHISTECH, Budapest, Hungary). For quantification of staining, positively stained cells were counted manually using the Cell Counter function of ImageJ.