Protease inhibitor cocktails and phosphatase inhibitors

Total proteins were extracted by RIPA Buffer (Beyotime) supplemented with protease inhibitor cocktails and phosphatase inhibitors (Sigma-Aldrich). Lysates were incubated at 100 °C for 5 min and centrifuged at 12,000 rpm for 15 min. After SDS-PAGE, samples were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). Subsequently, membranes were blocked in 5% skim milk at room temperature for 1 h and incubated at 4 °C overnight with antibodies raised against TBK1 (Abcam, ab40676), CXCR4 (Santa Cruz Biotechnology, sc53534), phosphoserine (Abclonal, AP0932), β-actin (Cell Signaling Technology, 3700), GFP (Proteintech, 50430), or His (Proteintech, 66005). Afterward, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. A chemiluminescence signal was detected using the WesternBright ECL (Advansta) with Image Lab software.

Total proteins were extracted by RIPA Buffer (Beyotime) supplemented with protease inhibitor cocktails and phosphatase inhibitors (Sigma-Aldrich). The appropriate primary antibodies were added to the supernatant of the lysate for rotation at 4 °C overnight. Then, resuspended protein A/G agarose was added and incubated at 4 °C for 4 h. After wash with RIPA Buffer, the immunoprecipitants were boiled in SDS-PAGE loading buffer, followed by western blot assays. The secondary antibodies were as follows: anti-mouse IgG LCS(Abbkine, A25012) and anti-rabbit IgG LCS (Abbkine, A25022).