Gapdh antibody

RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China) was used for the
extraction of cellular protein. Protein concentration was measured using the BCA
Protein Assay kit (Pierce Biotechnology, Rockford, IL, USA). Protein lysates
(10-30ug) were separated by 10% SDS-PAGE gel (Thermo Fisher Scientifc, Inc.) and
transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA,
USA). Membranes were blocked with nonfat milk (5%) and incubated with SOX4
antibody (1:2,000, ab85051; Abcam, Cambridge, UK), Vimentin antibody (1:1000,
#5741, Cell Signaling Technology, CA, USA), AKT antibody (1:1000, #4691, Cell
Signaling Technology), p-AKT antibody (1:1000, #4060, Cell Signaling Technology)
or GAPDH antibody (1:4,000, 5632 -1; Epitomics, Burlingame, CA, USA) at 4°C
overnight. Then membranes were washed with Tris buffered saline Tween (TBST)
solution and incubated with horseradish peroxidase-conjugated secondary
antibodies for 1 h at room temperature. ECL reagent was used for the
visualization and detection of protein signals.

At 24 h post-transfection, C666-1 and 13-9B cells were harvested, and 1×10⁵ cells were mixed with 1 ml RIPA solution (Sangon Biotech Co., Ltd.) to extract total proteins. The BCA method (Sigma-Aldrich; Merck KGaA) was used to measure protein concentration. Proteins were incubated with sample buffer and boiled for 5 min to denature the proteins. Protein samples (30 µg per lane) were subsequently loaded on a 12% gel, resolved using SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with PBS containing 5% skimmed milk at 22°C for 2 h. Membranes were incubated with rabbit polyclonal primary antibodies against PTEN (1:1,200; cat. no. ab31392; Abcam) or GAPDH antibody (1:900; cat. no. ab9485; Abcam) at 4°C overnight, and subsequently with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (1:1,200; cat. no. MBS435036; MyBioSource, Inc.) at 22°C for 2 h. Enhanced chemiluminescence reagent (Sigma-Aldrich; Merck KGaA) was used to detect the signal on the membrane. Densitometry analysis was performed using Image J version 1.46 (National Institutes of Health). GAPDH was used as the loading control.

Total protein was extracted from transfected cells and tissues using RIPA lysis buffer (Cat. No. P0013C, Beyotime, Beijing, China) according to the manufacturer’s instructions. For Western blots, equal amounts of protein were separated on 12% SDS polyacrylamide gels and transferred to nitrocellulose membranes (Cat. No. FFN02, Beyotime, Beijing, China). After proteins were transferred, the membrane was blocked with 5% nonfat milk for 2 hours and then incubated with a PTEN antibody (1:2000, Cat. No. ab32199, abcam, San Francisco, USA), a β-actin antibody (1:5000, Cat. No, ab16039, abcam, San Francisco, USA) and an HRP-conjugated GAPDH antibody (1:5000, Cat. No. HRP-60,004, Proteintech, Chicago, USA) overnight. After washing with Phosphate Buffered Saline with 0.05% TWEEN (PBST), membranes were incubated with a goat anti-rabbit HRP-conjugated secondary antibody (Cat. No. sc-2005, Santa Cruz Biotechnology, Dallas, USA) and visualized using enhanced chemiluminescence (Beyo ECL Star, Cat. No. P1008AS, Beyotime, Beijing, China). The expression levels of proteins were semiquantitatively analyzed with ImageJ v1.8.0 (National Institutes of Health). The GAPDH or β-actin signal was used as a loading control.

Annexin V-FITC, MTT kits, and HRP-labeled Goat Anti-Rabbit IgG (A0208) were from Beyotime Biotechnology; RPMI-1640 medium, fetal bovine serum, penicillin-streptomycin and trypsin from Gibco; Thermo Fisher Scientific, Inc.; TRIzol reagent and Transwell cell culture plates from Corning Inc.; Promega M-MLV reverse transcription kits from Promega Corporation; YBR Premix Ex Taq from Takara Biotechnology Co., Ltd.; miR-15a overexpression plasmid was synthesized by Guangzhou RiboBio Co., Ltd.; Lipofectamine^® 3000 Transfection kit was from Invitrogen; Thermo Fisher Scientific, Inc.; Smad3 protein (rabbit anti-human Smad3 monoclonal antibody, ab40854) from Abcam; GAPDH antibody (mouse anti-human GAPDH monoclonal antibody, SC-32233) from Santa Cruz Biotechnology, Inc.; Immobilon Western HRP from Thermo Fisher Scientific, Inc.
Human NSCLC cell lines (A549, H1299, and H1975) and the normal lung cells (BEAS-2B) were all from Shanghai Institute of Biochemistry and Cell Biology, CAS. The cells were cultured in DMEM (Corning Inc.) medium containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc.) and 1% streptomycin (Corning Inc.) at 37°C with the concentration of 5% CO₂.