Protein a g beads

To detect the interaction between exogenous VRK2 (Flag-VRK2) and IKKβ (HA-IKKβ), Flag-VRK2 and HA-IKKβ plasmids were transfected into 293T cells. 48 h after transfection, the cells were lysed with IP lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, protease inhibitor and phosphatase inhibitor), and the supernatant was collected. Beads coupled to an anti-Flag antibody (Sigma, A2220) were added to the supernatant for incubation overnight at 4°C. The next day, the beads were washed 3 times in wash buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1% NP-40), 1× loading buffer was added, and the beads were heated at 100°C for 5 min. Then, the supernatant was collected for western blot analysis.
To detect whether there was any interaction between endogenously expressed VRK2 and IKKβ in pancreatic cancer cells, IP lysis buffer containing protease and phosphatase inhibitors was used to lyse cells. Equal amounts of protein were aliquoted, and 0.25 μg of an anti-IKKβ antibody was added for incubation overnight at 4°C. The next day, 40 μL of Protein A/G beads (Bimake, B23202) was added for another incubation overnight at 4°C. The beads were washed 3 times with wash buffer, and 1× loading buffer was then added for western blot analysis.

Cells in each group were lysed in lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, 1 mM EDTA, 0.5 % NP-40 and protease inhibitor mixture] and centrifuged to move cell debris. After measurement of the protein concentration by BCA, the same amount of protein was taken from each group and made up to the same volume with cell lysate, followed by the incubation with 1 μg anti-Beclin 1 (ab207612, 1:30), IgG (ab172730, 1:100), and 15 μL protein A/G beads (B23201, Bimake, Houston, TX, USA) for 2 h. After washing the cell lysates three times, beads were collected by centrifugation, added with an equal volume of reducing sample buffer, and boiled at 100° C for 5 min. Samples were separated by SDS-PAGE and transferred to PVDF membranes (Millipore), followed by Western blot analysis.

For immunoprecipitation with mouse hypothalamus, protein was extracted from samples by mincing them with scissors on ice in immunoprecipitation (IP) buffer (50 mM Tris, 150 mM NaCl, 0.5% NP40, pH 7.5) containing protease inhibitor cocktail, and 1 mM PMSF. For immunoprecipitation with cultured PC12 cells to detect protein ubiquitinoylation, 24 h after the co-transfection of Myc-Ub and HA-PGC-1α plasmids, PA (100 μM) treatment was performed for 12 h, 24 h, and 36 h; then, the cell lysates were obtained by homogenizing cells in the same IP buffer mentioned above. Then, a centrifugation (14,000× g for 10 min, 4 °C) was performed to obtain the supernatant and the protein concentration was measured with BCA Protein Assay Kit (PC0020, Solarbio, Beijing, China) according to the manufacturer’s protocol. After the adjustment of protein concentration, every 200 μL of lysates were incubated overnight at 4 °C with 50 μL pretreated Protein A/G beads (B23202, Bimake, Beijing, China), which were first incubated with 5 mg/mL antibody for 1 h at 4 °C. The immunocomplex was washed three times with washing buffer (50 mM Tris, 150 mM NaCl, 0.5% detergent (NP40), pH 7.5) and resuspend using 1× loading buffer under 100 °C water bath heating for 10 mins. Finally, proteins were separated by SDS-PAGE and immunoblotted with indicated antibodies.

For western blotting analysis, cells were lysed in RIPA buffer (P0013B, Beyotime) containing protease and phosphatase Inhibitor cocktail, EDTA free (78441, Thermo Fisher Scientific) for 30 min on ice followed by pelleting of insoluble material by centrifugation. Protein concentration was determined using a BCA assay. For co-immunoprecipitation experiments, 10 million cells were lysed in 1 ml NP40 buffer (P0013F, Beyotime) containing protease and phosphatase Inhibitor cocktail, EDTA free, for 30 min on ice. After concentration, supernatants were incubated with Rabbit anti-DENR, Rabbit anti-JAK2, or Rabbit normal IgG for 4 h at 4 °C, followed by addition of 25 µl protein A/G beads (B23201, Bimake) and incubation for a further 2 h. After five washes in NP40 buffer, beads were eluted in 100 μl IgG elution buffer, PH2.0 (21028, Thermo Fisher Scientific) and neutralized with 15 μl 50 mM NaOH.

Total proteins were extracted by NP-40 buffer as mentioned above. The concentration of proteins was measured, and the samples were respectively divided into three parts (Input, IgG, IP). Each part contained about 1-2 mg protein. 20 μl protein A/G beads (#B23201, Bimake, USA) were added to each of the sample parts and the mixture was incubated for 2 h at 4℃. Then, IgG or IP antibodies were added to the samples, and the mixture was incubated overnight at 4℃. The next day, 20 μl protein A/G beads were added to the samples again and incubated together for 2 h at 4℃. Magnetic beads were collected, mixed with NP40 lysis and loading buffer, and denatured at 95℃ for 5 min. Then the supernatant was collected by centrifugation. At last, protein-protein interaction was detected by western blotting as mentioned above.

Immunoprecipitation and nuclear co-precipitation were performed according to the manufacturer’s instructions (Protein A/G beads, #B23201, bimake, Shanghai, China). The details are provided in SI Materials and Methods.

The cells were harvested and washed twice with ice-cold PBS buffer. Cells were then sonicated in IP buffer [20 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na₃VO₄, and protease inhibitor cocktail (Bimake), pH 7.5] at 4 °C for 3 times per 5 s by Bio-ruptor UCD-200 (Diagenode, Belgium), followed by centrifuged at 13,300 g at 4 °C for 10 min to remove the cell debris. Expressing of the indicated proteins in the lysates was checked by western blotting using relevant antibodies to normalize total amounts of the inputs. The supernatants were each incubated with specific antibodies or normal IgG (as control), and equal amounts of protein A/G beads (Bimake) overnight at 4 °C. The protein A/G beads with the bound antibodies and interacting proteins were pelleted and washed 3 times with IP buffer before boiled in 2× SDS-PAGE sample. The boiled samples were then resolved in SDS-PAGE and subject to western blot analysis.