Sample buffer

hiHeps were harvested and lysed with 100 μL protein extraction reagent RIPA lysis buffer (Beyotime, China) containing 10 mmol/L phenylmethylsulfonyl fluoride (PMSF, Beyotime, China) on ice for 30 minutes. The supernatant of each sample was collected by centrifugation at 12,000 rpm for 30 minutes at 4°C, and protein concentrations were quantified using a BCA assay (Beyotime, China), following the manufacturer’s instructions. After being heated for 5 minutes at 95°C in sample buffer (Beyotime, China), protein electrophoresis was performed with 10% SDS‐PAGE gels. Proteins were transferred to PVDF membranes (Millipore) at 100 V for 100 min using a blotting apparatus. Membranes were blocked with 5% (w/v) non‐fat dry milk in TBST buffer (20 mmol/L Tris‐HCl, pH 7.4, 150 mmol/L NaCl, 0.05% Tween‐20) for 30 minutes at room temperature and incubated with primary antibodies (Table 2) overnight at 4°C . Then, the membranes were washed with PBST for 5 minutes for a total of five times and were detected with the appropriate secondary antibodies conjugated with horseradish peroxidase (HRP) (1:10,000, Abcam) at room temperature for 1 hour and were visualized by enhanced chemiluminescence (Millipore). β‐actin served as the internal control, and the results of the Western blotting were analysed by calculating the gray value.

Protein samples were obtained from cells after treatment with radio-immunoprecipitation assay lysis solution (Beyotime, Shanghai, China) and then quantified by the bicinchoninic acid kit (Beyotime). The proteins were mixed with the sample buffer (Beyotime), denatured, and electrophoresed at 80 V. After transferring onto a membrane, this latter was blocked in the blocking solution, incubated with the primary antibodies, HIF-1α (1:6000, ab205718), hexokinase 2 (HK2, 1:1000, ab209847), LDHA (1:5000, ab52488), GLUT1 (1:100000, ab115730, (Abcam, MA, USA), and then washed in PBS and incubated with secondary horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG) antibody (1:5000, CWBIO, Beijing, China). The membrane was developed, and protein bands were visualized using a chemiluminescence imaging system (Bio-rad, CA, USA).

Cultured cells or kidney tissues were lysed in sample buffer (Beyotime, China) and protein concentration was determined using Nanodrop 1000 Spectrophotometer (Thermo, USA). Protein samples were analyzed by Western blot as previously described30 (link). Cells in logarithmic phase were seeded at the density of 60 ~ 70% confluence per well into 24-well chamber slides. After treatment with test samples for the indicated times, cells were analyzed by immunofluorescence staining as previously described30 (link). Cultured SV40-Mes13 cells or kidney tissues were lysed in TRIZOL reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated. Then RNA was analyzed by RT-PCR as previously described30 (link). Primers used for the reactions were purchased from Genscript and the primer sequences are listed as followed: MCP-1(Forward: 5'-AGGTGTCCCAAAGAAGCTGTA-3', Reverse: 5'-ATGTCTGGACCCATTCCTTCT-3'), CSF-1(Forward: 5'-CCCATATTGCGACACCGAA-3', Reverse: 5'-AAGCAGTAACTGAGCAACGGG-3'), ICAM-1(Forward: 5'-GCCTTGGTAGAGGTGACTGAG-3', Reverse: 5'-GACCGGAGCTGAAAAGTTGTA-3'), VCAM-1(Forward: 5'-TGCCGAGCTAAATTACACATTG-3', Reverse: 5'-CCTTGTGGAGGGATGTACAGA-3').

The protein concentration in the Ad5-GFP and EVM/VSV-G Ad5-GFP virus suspensions was measured after purification via density gradient centrifugation using the BCA method, and 80 μl of the suspension was mixed with 20 μl of 5× sample buffer (containing β-mercaptoethanol) (Beyotime, Hangzhou). Samples were incubated at 100°C for 5 min. Samples were separated on 12% SDS-PAGE gels and electrotransferred onto a polyvinylidene fluoride membrane (#03010040001; Roche). The membrane was blocked with TBS containing 10% non-fat milk for 30 min and then probed with a primary anti-VSV-G tag antibody (1:2,000, ab50549 Abcam, Cambridge, MA, United States) anti-CD63 antibody (1:1,000, ab59479 Abcam, Cambridge, MA, United States) anti-CD9 (1:1,000, 13403 Cell Signaling Technology, Danvers, MA, United States) and then a secondary horseradish peroxidase-labeled rabbit anti-mouse monoclonal antibody (1:5,000, A00160 GenScript Biotech Corp; Nanjing, China). The membrane was exposed to chemiluminescence reagent (WBKLS0500; Millipore, Billerica, MA, United States), and images were captured using a chemiluminescence imaging apparatus (ChampChemi 610; Sage Creation Science, Beijing, China).