Cw0098m

For co-immunoprecipitation assays, HEK293T cells were harvested and lysed with either TNE lysis buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA and 0.5% Nonidet P-40] or RIPA buffer (R0010, Solarbio) containing a protease inhibitor mixture (1697498001, Roche). Lysates were incubated with anti-Flag-agarose beads (A2220, Sigma-Aldrich) or protein A-Sepharose beads (101041, Invitrogen) at 4°C for 4 h. Beads were washed four times with TNE or RIPA buffer, and bound proteins were then separated by SDS-PAGE and visualized using western blots.
For immunoblotting experiments, we used the following affinity-purified antibodies: anti-Flag (1:1000; Cell Signaling Technology, 2368S), anti-Myc (1:3000; M047-3, MBL), anti-HA (1:3000; CW0092A, CW), anti-β-tubulin (1:5000, CW0098M, CWBIO), anti-p-Thr (1:300; ab9337, Abcam) and anti-p-Ser (1:250; ab9332, Abcam).

Western blotting analyses were performed as described previously (Wu et al., 2016 (link); Zhou et al., 2017 (link)). The following antibodies were used: rabbit monoclonal anti-TSPO (ab109497, Abcam, Cambridge, MA, USA), rabbit monoclonal anti-iNOS (D6B6S, Cell Signaling Technology, Cambridge, MA, USA), mouse monoclonal anti-β-actin (66009-1-Ig, Proteintech, Wuhan, China), mouse monoclonal anti-β-tubulin (CW0098M, CWBiotech, Beijing, China), and rabbit monoclonal anti-arginase-1 (93668S, Cell Signaling Technology).

For immunoblotting experiments, we used the following affinity-purified antibodies: anti-Flag (1:5,000; F2555, Sigma-Aldrich), anti-Myc (1:3,000; M047-3, MBL Medical & Biological Laboratories, Nagoya, Japan), anti-HA (1:3,000; CW0092A, CWBIO, Beijing, China), anti-β-Tubulin (1:5,000, CW0098M, CWBIO), and anti-Foxj1 (1:200; ab220028, Abcam).
For coimmunoprecipitation assays, embryos or HEK293T cells were harvested and lysed with TNE lysis buffer (10mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM EDTA, and 0.5% Nonidet P-40) containing a protease inhibitor mixture. Lysates were incubated with anti-Flag-agarose beads (A2220, Sigma-Aldrich) or protein A-Sepharose beads (101041, Invitrogen) and anti-phospho-threonine–proline antibody (1:5,000; 9391, Cell Signaling Technologies) at 4 °C for 4 hours. Beads were washed 4 times with TNE buffer. Bound proteins were then separated by SDS-PAGE and visualized by western blots.