Ab34710

The expression of ULK1 and LC3 protein was detected by Western blot. Total protein was extracted from cardiac fibroblasts by the Western & IP cell lysis buffer (Beyotime, China) and protein concentration was determined by BCA method. For Western blotting, equal amounts of protein samples (50 μg) were separated by sodium dodecy sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (AmershamTMHybondTM, Germany). The PVDF membrane was blocked with 5% bovine serum album (amresco, USA) for 1 h and then incubated with primary antibody at 4 °C overnight. Primary antibodies were: rabbit anti-ULK1 (Abcam, ab128859) diluted 1:1000; rabbit anti-LC3 (Cell Signaling, #4108) diluted 1:1000; rabbit anti-Collagen I (Abcam, ab34710) diluted 1:1000; rabbit anti-GAPDH (Cell Signaling, #5174) diluted 1:1000. On the next day, the membranes were washed with TBS-T (0.1% Tween-20) and further incubated with HRP-linked secondary antibody (Cell Signaling, #7074) diluted 1:2000. The membranes were then visualized by chemiluminescence BeyoECL Plus (Beyotime, China).

RIPA buffer (CWBIO, China) supplemented with protease inhibitors and phosphatase inhibitors (CWBIO, China) was used to extracted protein from kidney tissues or HK-2 cells. Protein concentration was quantified by the BCA method. Equal amounts of protein were then analyzed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. Primary antibodies specific for AdipoR1 (1:3000, Abcam, ab126611), phospho-AMPKα(Thr172) (1:1000, Cell Signaling Technology, 2535), AMPKα1 + AMPKα2 (1:2000, Abcam, ab131512), phospho-ULK1 (Ser555) (1:1000, Cell Signaling Technology, 5869), ULK1 (1:1000, Cell Signaling Technology, 8054), ADRP (1:500, Abcam, ab52356), FN (1:1000, Abcam, ab2413), Collagen I (1:1000, Abcam, ab34710), LC3B (1:1000, Cell Signaling Technology, 3868), Beclin1 (1:800, Proteintech, 11306-1-AP), ATG5 (1:1000, Proteintech, 10181-2-AP), SQSTM1/ P62 (1:1000, Proteintech, 18420-1-AP, Cell Signaling Technology, 1:1000, 5114) and TFEB (1:1000, Proteintech, 13372-1-AP) were used. The bands were evaluated using a Tanon 5200 Multi instrument (Tanon Instruments, China). The band densities of target proteins were compared with those of β-actin (1:5000, Proteintech, 60008-1-Ig), GAPDH (1:5000, Proteintech, 60004-1-Ig) or Lamin B (1:1000, Proteintech, 66095-1-Ig) by using densitometry software (ImageJ).

N-[(1R)-1,2,3,4-Tetrahydro-1-naphthalenyl]-1H-benzimidazol-2-amine hydrochloride (NS-8593), apamin, human transforming growth factor (TGF-β1; T7039), plasminogen, α2-antiplasmin and D-Val-Leu-Lys-7-amido-4-methylcoumarin were all from SigmaAldrich. Waixenicin A was isolated as described previously (Zierler et al. 2011 (link)). For protein detection specific antibodies against PAI-1 (abcam, ab66705), fibronectin (abcam, ab2413), smooth muscle actin (abcam, ab5694), collagen-1 (abcam, ab34710), p-SMAD-2 (cell signaling, clone 138D4, #3108), SMAD-3 (cell signaling, clone C67H9, #9523), SMAD-2 (cell signaling, clone D43B4, #5339), SDHA (abcam, ab14715) and histone H3 (abcam, ab1791) were used.