Gapdh 60004 1 1g

Proteins (20–50 µg) were boiled for 5 min in SDS sample buffer (62.5 mM Tris (pH 6.7), 1.25% SDS, 12.5% glycerol, and 2.5% β-mercaptoethanol). Proteins isolated from the cells and HR Pre-Stained Protein Marker 10–170 kDa (BIOTOOLS, New Taipei City, Taiwan) were loaded onto SDS gels and subjected to electrophoresis. After transfer to a PVDF membrane, the proteins on the membrane were incubated with antibodies against Beclin 1 (3738, Cell signaling, Beverly, MA, USA), LC3 (4108, Cell signaling), collagen 1 (14695-1-AP, Proteintech, Rosemont IL, USA), CTGF (23936-1-AP, Proteintech), PAI-1 (11907, Cell signaling) and GAPDH (60004-1-1 g, Proteintech). Immunoreactive bands were visualized using an enhanced chemiluminescence system (Amersham, Little Chalfont, United Kingdom). Protein expression was quantified by ImageQuant version 5.1 (GE Healthcare, Waukesha, WI, USA).

The following antibodies were used in this study: FBXO2 (sc-398111, Santa Cruz), EBNA1 (ab81581, Abcam), Zta (sc-53904, Santa Cruz), β-tubulin (sc-23948, Santa Cruz), Myc (sc-40, Santa Cruz), GAPDH (60004-1-1g, Protein-tech) and FLAG (F3165, Sigma). MG132, bafilomycin A1 and tunicamycin were purchased from Selleckchem, LLC. siRNAs were synthesized by Guangzhou RiboBio Co., Ltd. The siRNAs were 21 base pairs long, and the sequences were as follows: control siRNA: UUCAAUAAAUUCUUGAGGUdTdT; FBXO2 siRNAs: #1: GAUGAGAGCGUCAAGAAGUdTdT; #2: CAGUUCUACUUCCUGAGCAdTdT; #3: AAGGUAGAUAGGCCUUAACdTdT. Lipofectamine RNAiMAX (Invitrogen) was used for siRNA transfection.

Protein concentrations were initially measured using the BCA method (Thermo Fisher Scientific). Thereafter, total protein (25 μg/well) was fractionated on 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred overnight to nitrocellulose membranes (Millipore, Billerica, MA, USA), which were then incubated sequentially with 5% nonfat dry milk at room temperature for 1 hr, primary antibodies at 4°C overnight, and then secondary antibody anti-mouse IgG (1 : 1,000; Beyotime, Shanghai, P.R. China) at 37°C for 1 hr. An enhanced chemiluminescence system (Tanon, Shanghai, P.R. China) was used to determine the protein levels. The primary antibodies used included the following: PCSK6 (Ab151562, Abcam, UK), CD63 (25682-1-AP, Proteintech, USA), CD81 (66866-1-Ig, Proteintech, USA), and GAPDH (60004-1-1 G, Proteintech, USA).

Total protein of samples was extracted using RIPA lysis buffer (Beyotime, China). The protein was fractionated on SDS-PAGE and transferred to nitrocellulose membrane (Millipore, USA). The primary antibody was applied at 4 ℃ overnight. The protein was detected by ECL after the application of secondary antibody. All primary antibodies used in this study were list as follows: TRIM14 (15742-1-AP, Proteintech, USA); Cleaved-caspase3 (Ab2302, Abcam, UK); AKT (46191, CST, USA); p-AKT (4060, CST, USA); CD9 (Ab92726, Abcam, UK); CD63 (Ab271286, Abcam, UK); CD81(Ab109201, Abcam, UK); GAPDH (60004-1-1G, Proteintech, USA). Three replications were needed for each samples and three independent experiments for each reaction Cell transfection.
To knockdown the expression of TRIM14, three short interfering RNAs (siRNAs) were generated (Major, China) and constructed into lentiviral plasmids. A negative control siRNA (siNC) was used as the control group. The sequence of three siRNAs were as follows: siTRIM14-1: 5’- GCAGCACATTGACAACATA -3’; siTRIM14-2, 5’- GCCCGTCAAGAGCTTCTTT-3’; siTRIM14-3 5’- GCGATCGCTATTGCTGAAA-3’. For overexpression, a lentiviral plasmid containing TRIM14 cDNA was generated with a mock plasmid as a negative control (oeNC). Lipofectamine 2000 (Invitrogen, USA) facilitated the cell transfection.