Membrane protein extraction kit

4T1 cell membranes were extracted using the Membrane Protein Extraction Kit according to the instructions provided by Beyotime Biotechnology. Briefly, 4T1 cells were incubated in cell culture dishes with diameter of 15 cm, and then the cells (1 × 10⁸) were collected by a cell scraper and centrifuged at 700×g for 5 min. The cell precipitation was resuspended in precooled PBS buffer (pH = 7.4) followed by centrifugation at 600×g for 5 min. In order to remove the residual PBS buffer, further centrifugation for 1 min was performed. The obtained cell pellets were suspended in Membrane Protein Extraction solution (3 mL), and phenylmethanesulfonyl fluoride (PMSF, 1 × 10^–3 M) was added. After that, the cells were incubated in an ice bath for 15 min. Thereafter, repeated freeze-thawing was carried out to break the cells in the above solution and then centrifuged at 700×g for 10 min at 4 ℃. The collected supernatant was further centrifuged at 14,000×g for 30 min to collect the CCMs. The CCMs was collected and lyophilized for later use [48 (link), 57 (link)]. All cell membrane extraction processes were carried out in an ice bath.

Membrane protein of liver samples was extracted with membrane protein extraction kit (Beyotime Institute of Biotechnology, China) according to the protocol. Protein concentrations were measured with a modified BCA technique. An equal amount of membrane protein (100 μg) per lane was separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the gels were transferred to polyvinylidene difluoride membranes which were blocked with Tris-buffered saline containing 5% nonfat milk. Then the membranes were incubated overnight at 4°C in Tris-buffered saline containing 0.1% Tween 20 (TBST), 5% nonfat milk, and anti-PDZK1 (at the dilution of 1 : 200). After being washed three times in TBST, the membranes were incubated with HRP-conjugated secondary antibody for 2 h at room temperature and subsequently processed for enhanced chemiluminescence (ECL) detection using potent ECL kit (Multisciences, China). Signals were detected using a chemiluminescence detection system (IS4000MM Pro, Kodak, USA). β-Actin was used as a loading control.

Before the adipocytes from different groups were lysed in RIPA buffer, the cells were stimulated by 100 nM insulin for 10 min. After that, the samples were then centrifuged (4 °C, 12,000 rpm, 15 min) and the supernatants were collected. The separation of membrane proteins from the cells was performed refer to the directions prepared by the manufacturer of the membrane protein extraction kit (Beyotime Biotechnology, Shanghai, China). The concentration of the protein in the supernatant was determined using BCA protein assay reagent (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein (30–50 μg) in different groups were electrophoresed in SDS-PAGE gel, and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked by 5% non-fat milk powder in TBS-T buffer for 2 h at room temperature, and then incubated with various primary antibodies for overnight at 4 °C. After washing with TBS-T buffer, the PVDF membranes were incubated with appropriate secondary antibodies (dilute 1:10,000) for 2 h at room temperature. Protein bands were detected by enhanced chemiluminescence method using the ECL kit. The protein bolts were visualized by autoradiography, and the bolts was quantified via a Gel Pro analyzer. Normalization of protein expression was carried out using β-actin.

The treated cells were washed with PBS and lysed in a RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with a protease inhibitor cocktail (Boster, Wuhan, China) to extract total proteins. The membrane protein was extracted from the cells using a Membrane Protein Extraction Kit (Beyotime, Shanghai, China). Immunoblotting analysis was performed as previously described (Yin et al., 2016 (link)) following standard procedures and using the primary antibodies, ABCA1, LDLR (Abcam), LXR (ABclonal), and Anti-β-actin (ProteinTech). In brief, the protein was separated on an 8–10% SDS denatured polyacrylamide gel and then transferred onto a NC membrane. The membranes were blocked with 5% skim milk and were incubated with appropriate antibodies at 4°C overnight. Then, the membranes were washed and incubated with horseradish peroxidase–conjugated secondary antibodies (1:3,000; Sanjian, Tianjin, China). The protein of interest was visualized using an Immobilon Western Chemiluminescent HRP Substrate (Thermo Fisher Scientific).