Protein a g agarose beads

Fibroblasts were washed twice, harvested in PBS, and then lyzed in ice-cold IP lysis buffer containing 50 mM Tris, 1 mM EDTA, 0.5% NP-40, 150 mM NaCl, 10% glycerol and 1 mM PMSF for 30 min. Total cell extracts were centrifuged for 30 min at 12,000 rpm at 4 °C, then the protein A/G agarose beads (Beyotime Bio, Shanghai, China) or control IgG (Santa Cruz) was incubated with the supernatant for 1 h as a pretreatment. Lysates were incubated overnight with anti-PARK2 monoclonal antibody (Abcam, No. ab179812), anti-BECN1 monoclonal antibody (Abcam, No. ab207612) or control IgG and protein A/G agarose beads on rotary shaker at 4 °C. The immunoprecipitated complexes were collected by centrifugation at 3000 rpm for 5 min at 4 °C, and the beads were washed three times with IP lysis buffer and resuspended in 2 × SDS loading buffer. Subsequently, the immunoprecipitates complexes were eluted from the beads at 99 °C for 5 min. The eluted proteins were determined by Western blot analysis with indicated antibodies.

Cells were collected and lysed using RIPA lysis buffer (Beyotime Biotechnology) containing protease inhibitors for 30 min at 4°C. The protein was incubated with anti-G6PD and anti-IgG antibodies at 4°C overnight. Then Protein A/G agarose beads (Abcam) was added to each tube. After incubation again at 4°C for 3 h, the beads were collected by centrifugation at 2,000 x g for 2 min. Subsequently, the beads were washed with IP wash buffer three times, followed by adding 40 μL 2× loading buffer and denaturing at 100°C for 5 min. Western blot was then performed. The anti-O-GlcNAC antibody (RL2, Thermo Scientific) was used to test the O-GlcNAcylation of G6PD.

Co-immunoprecipitation (co-IP) was performed as previously described (Yan et al., 2017 (link)). The cells were washed once with cold PBS, lysed in lysis buffer for IP (Beyotime, #P0013) containing a protease inhibitor cocktail (Roche) at 4°C for 1 h, and then centrifuged at 12,000 g for 15 min at 4°C. After centrifugation, a small fraction of the supernatant was frozen for subsequent analysis (cell extract) as the input. The remaining fraction was incubated at 4°C with 1 μg of the desired antibody on a rotator overnight. Then, the mixtures were incubated with Protein A/G agarose beads (Abcam, #ab193262) with rotation at 4°C for 6 h. After the incubation, the beads were washed three times with wash buffer, boiled in loading buffer at 95°C, and then subjected to western blotting. The following specific secondary antibodies were used to avoid overlap of the bands for the target proteins with those of the immunoglobulin light chain and heavy chain: IPKine HRP Goat AntiMouse IgG HCS (Abbkine, #A25112, 1:5,000), IPKine HRP Goat AntiRabbit IgG HCS (Abbkine, #A25222, 1:5,000), IPKine HRP AffiniPure Goat AntiMouse IgG Light Chain (Abbkine, #A25012, 1:5,000), and IPKine HRP AffiniPure Mouse AntiRabbit IgG Light Chain (Abbkine, #A25022, 1:5,000).

Co-IP analysis was used to confirm MBL can inhibiting LOX1-ox-LDL binding. The treated cells were collected, and proteins were extracted. An ultraviolet spectrophotometer was used to determine the protein concentration. Then, 20 μL of Protein A/G agarose beads, 180 μL of PBS, and MBL antibody at a ratio of 1:100 (Abcam, Cambridge, UK) was mixed and incubated at room temperature for 1.5 h with rotation. Samples were centrifuged at 4 °C for 2 min at 2000 rpm/min and washed with PBS 3 times, after which the 400 μL (1 mg) protein lysate was added, and the samples were rotated and incubated at 4 °C for 2 h. After centrifugation at 2000 rpm/min at 4 °C for 2 min, they were washed with PBS 3 times, then the precipitation was collected and added into 1 × Loading Buffer and boiled for 5 min. The supernatant was collected after centrifugation at 12,000 r/min at 4 °C for 5 min and then prepared for the Western Blot experiment (see Section 2.9).