Protein a g agarose

Melanoma cells were lysed in NP-40 lysis buffer (Beyotime, China) containing protease and phosphatase inhibitors (Selleck, USA). To block nonspecific protein binding, the cell lysates were first incubated with a normal IgG antibody (CST, 2729#, USA) or protein A/G-agarose (Beyotime, China) for 1 hour at 4 °C. The beads were discarded, and the supernatant was retained. The supernatant was incubated with a normal IgG antibody or anti-TET3 (1:1 000, CST) and protein A/G-agarose (Beyotime, China) for 4 h at 4 °C. Then, the beads were washed 3 times with NP-40 buffer and loaded to 8% or 10% SDS–PAGE for immunoblotting.

Worms (9cm plates x 10) were collected and washed with M9 buffer. The worm pellet was lysed by French Press in ice-cold lysis buffer (25 mM Tris-HCl pH 7.5, 100 mM NaCl,1 mM EDTA, 0.5% NP-40, 1 mM PMSF, 1 mM Na₃VO₄, 1 μg/ml Pepstatin-A and 10 mM NaF) containing protease-inhibitor cocktail (Sigma, St. Louis, MO). The lysates were incubated at 4°C for 30 min and centrifugated at 13,000xg for 30min. Then, supernatant was incubated with 80μl Protein A+G Agarose (Beyotime, Shanghai, China) for 1h at 4°C to pre-clear non-specific bead-protein interactions. 2μl anti-GFP antibody (ab290) was added into pre-cleared supernatant and incubated at 4°C overnight, followed by incubation with 80μl Protein A+G Agarose (Beyotime, Shanghai, China) at 4°C for 4 hours. Precipitates were washed five times with lysis buffer and subjected to immunoblotting using anti-actin and anti-GFP polyclonal antibodies.

A Co-IP assay was conducted as described previously [31 (link)]. After 24 hours of treatment with Mn, brain slices were rinsed three times in ice-cold PBS buffer, followed by incubation for 30 min at 4°C with 1 ml ice-cold IP lysis buffer (Beyotime, Haimen, China) containing the serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Samples were pre-cleared with Protein A+G agarose (Beyotime). Pre-cleared lysates were then incubated with mouse anti-full-length alpha-synuclein monoclonal antibody (1:50) or rabbit anti-calpain 1 polyclonal antibody (1:50) at 4°C overnight. A 25% slurry of Protein A+G agarose was added into the lysates incubated for 2 h at 4°C and washed with ice-cold IP lysis buffer (Beyotime). The pellet was resuspended in SDS loading buffer, boiled for 10 min, and then centrifuged at 12,000 × g for 1 min in a Sigma 3K 30 centrifuge (Sigma, Germany). The supernatant was removed and loaded onto a 12% SDS-PAGE gel separated by electrophoresis and transferred onto a PVDF membrane. Membrane blots were probed with rabbit anti-calpain 1 polyclonal antibody or mouse anti-full-length alpha-synuclein monoclonal antibody and visualized by chemiluminescent detection reagents (Pierce).

Lung tissues were lysed in RIPA buffer. Protein A + G agarose (P2055-10 mL, Beyotime, Shanghai, China) was used for immunoprecipitation according to the manufacturer’s instructions.

Total protein (500 μg) was incubated with anti-CRMP2 (1:50; Abcam, ab129082) or anti-α-tubulin (1:50) (Abcam, ab7291) overnight at 4°C. An equal amount of protein was incubated with mouse IgG (Beyotime, A7028) or rabbit IgG (Beyotime, A7016) as negative controls. The protein samples were loaded as the input control. Protein A + G agarose (Beyotime, P2012) was washed with PBS three times and mixed with the samples. The mixtures were incubated at 4°C for 2 h. The beads were then washed with PBS three times. The immunoprecipitates were subsequently subjected to 12% SDS-PAGE and analyzed via western blot using anti-α-tubulin (1:5,000) or anti-CRMP2 (1:20,000) antibodies. The protein bands were visualized using an ECL chemiluminescent detection kit (Beyotime, P0018S).

Nuclear proteins from fetal lungs of each group or MLE12 cells from each treatment group were purified using the NE‐PER Nuclear and Cytoplasmic Extraction Reagents (#78835; Thermo Fisher Scientific). FOXA2 was immunoprecipitated using an anti‐FOXA2 antibody (sc‐6554; Santa Cruz Biotechnology) immobilized on protein A/G Agarose (Beyotime Institute of Biotechnology). Phosphorylated FOXA2 (p‐FOXA2) was analyzed by western blot using antibody against phospho‐threonine (SC‐5267; Santa Cruz Biotechnology).