Gapdh 60004 1 ig

Protein expression of renal tissues or cells was analyzed by Western blotting following a regular procedure. The primary and secondary antibodies used were as following: KLOTHO (A12028), γH2AX (phosphorylated histone H2AX, AP0099; Abclonal); NRF2 (sc‐722), α‐SMA (sc‐32251; Santa Cruz Biotech); DNMT1(ab188453), DNMT3b (ab79822; Abcam), DNMT3a (bs‐0497R; Bioss); GAPDH (60004–1‐Ig; Proteintech); goat anti‐rabbit IgG‐HRP and goat anti‐mouse IgG‐HRP (YFSA02 and YFSA01; Yifeixue Biotech).

Antibodies against the following proteins were used in this study: Bip (3177 s, Cell Signaling Technology [CST]), eIF2α (5324P, CST), Phospho-eIF2α (3398P, CST), SAPK/JNK (9252S, CST), Phospho-SAPK/JNK (9255S, CST), Caspase-12(2202S, CST), Cleaved Caspase-3 (9661S, CST), Cleaved PARP (9541S, CST), Phospho-EGFR and EGFR (11862S, CST), GAPDH (60,004–1-Ig, Proteintech). PE anti-human EGFR Antibody (352,904, Biolegend). The kinase inhibitor library was purchased from Shanghai Topscience Co., Ltd (Shanghai, China). Afatinib (HY-10261) were purchased from MCE.

The primary antibodies against fibronectin (FN, ab2413), type IV collagen (Col-IV, ab6586), quinolinate phosphoribosyltransferase (QPRT, ab171944, for cell Western blotting), nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1, ab45652, for kidney tissue Western blotting and immunofluorescence staining) and nicotinamide riboside kinase 1 (NRK1, ab169548, for cell Western blotting) were purchased from Abcam (Cambridge, MA, USA). Antibodies of anti-vimentin (#5741), anti-α-Tubulin (#3873) and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (#7074) were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against α-smooth muscle actin (α-SMA, A5228), β-actin (A5441) and QPRT (SAB1410425, for kidney tissue Western blotting and immunofluorescence staining) were from Sigma-Aldrich (St Louis, MO, USA). The anti-NMNAT1 (sc-271557, for cell Western blotting) and anti-NRK1 (sc-398852, for kidney tissue Western blotting and immunofluorescence staining) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against nicotinic acid phosphoribosyltransferase 1 (NAPRT1, 13549-1-AP), nicotinamide phosphoribosyltransferase (NAMPT, 11776-1-AP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 60004-1-IG) and HRP-conjugated anti-mouse IgG (SA00001-1) were purchased from Proteintech Group (Wuhan, China).

All proteins were extracted from transfected cell lines using the RIPA lysis buffer and relative concentrations were quantified with a NanoDrop 2000 spectrophotometer. Briefly, the cell lysates were electrophoresed with 12% SDS-polyacrylamide gels (P0012AC, Beyotime) and the targeted bands were fully transferred onto polyvinylidene difluoride (IPVH00010, Merck Millipore) membranes; after blocked with 5% fat-free milk, the membranes were incubated with primary antibodies, followed by incubation with secondary antibodies and observation under chemiluminescence system. The detailed procedure can be referred to our previous study [42 (link)]. All experiments were carried out with three replicates. Primary antibodies used were as follows: ATG5 (sc-133158, Santa Cruz), ATG7 (sc-376212, Santa Cruz), MAP1LC3B (LC3B, NB100–2220, Novus), and GAPDH (60004-1-Ig, Proteintech). The intensity of each indicated protein band was quantified by measuring its grayscale via using the Image J software (version 1.8.0, NIH, USA). The targeted protein bands were first normalized to the reference (GAPDH) and then compared with the first band in the same row, respectively. Finally, statistical analysis was performed in each group (dimethyl sulfoxide, CQ, and BafA1), respectively.

The commercially available antibodies used are as follows: KIF4A (sc-365145,Santa Cruz), cleaved-caspase-3 (#9915, Cell Signaling Technology), cleaved-caspase-7 (#8438, Cell Signaling Technology), cleaved-poly ADP-ribose polymerase (PARP, #5625, Cell Signaling Technology), Bcl-2 (#4223, Cell Signaling Technology), Bax (#5023, Cell Signaling Technology), Akt (pan) (#4691, Cell Signaling Technology), p-Akt (ser473) (#4060, Cell Signaling Technology), p-Akt (Thr308) (#13038, Cell Signaling Technology) and Skp2 (#2652s, Cell Signaling Technology), CDC20 (10252-1-AP, Proteintech), cyclin B1 (#4138, Cell Signaling Technology), α-Tubulin (66031-1-Ig, Proteintech), GAPDH (60004-1-Ig, Proteintech) and Ki67 (MA5-14520, Rochford).

Western blotting was performed as previously described [23 (link)]. Membranes were probed with the following antibodies: DDX18 (DF9258, Affinity Biosciences, Changzhou, China), CDK4 (AF2515, Beyotime, Shanghai, China), CDK6 (AG1575, Beyotime), CyclinD1 (ab32053, Abcam, Cambridge, MA, USA), BAX (ab32503, Abcam), P21 (ab109520, Abcam). All primary antibodies were diluted in Tris-buffered saline containing Tween 20 and 3% bovine serum albumin (BSA), and a peroxidase conjugate (Bioworld, Saint Louis Park, MN, USA) was used as the secondary antibody. GAPDH (60004-1-Ig, Proteintech, Rosemont, IL, USA) and β-ACTIN (20536-I, Proteintech) were used as a loading control. Immunoreactive bands were visualized using an enhanced chemo-luminescence kit (Thermo Fisher Scientific), and images were captured using a Amersham^TM ImageQuant^TM 800 molecular imager (Cytiva, Washington, DC, USA).

Human HCC cell lines PLC/PRF/5 and Huh7 were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. HCC-LM3 cells were taken from our laboratory stocks. All the HCC cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Corning, Inc., Corning, NY, USA) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, and were maintained in an incubator with 5% CO₂ at 37 °C. Sorafenib and Bafilomycin A1 were obtained from Selleck Chemicals (Houston, TX, USA). Primary antibodies used in this study were listed below: MTX1 (15529-1-AP, Proteintech, Wuhan, China), CISD1 (16006-1-AP, Proteintech, Wuhan, China), Beclin1 (11306-1-AP, Proteintech, Wuhan, China), LC3 (14600-1-AP, Proteintech, Wuhan, China), GAPDH (60004-1-Ig, Proteintech, Wuhan, China), β-Actin (sc-8432, Santa Cruz Biotechnology, USA) and FLAG (abs830005, Absin, China).