Mouse anti α tubulin

For Co-IP assays, HEK-293T cells were transfected as indicated: 36 h later, cells were lysed in IP buffer (1% Triton X-100, 150 mM NaCl, and 50 mM Tris/HCl (pH 7.5), 1 mM PMSF, and protease inhibitor cocktail) and incubated with anti-Flag M2 affinity gels for 4 h at 4 °C. For reverse IP, HEK-293T cells were lysed and incubated with anti-HA antibody and protein A/G agarose for 12 h at 4 °C. Agarose was washed five times with IP assay buffer, boiled in SDS sample buffer, and subjected to Western blot analysis. Western blotting and immunofluorescence (IF) were performed as previously [40 (link)].
The following antibodies were used: mouse anti-Flag (F3165, Sigma-Aldrich), rabbit anti-Flag (F7425, Sigma-Aldrich), rabbit anti-GST (2622, Cell Signaling Technology), mouse anti-β-actin (66009-1-Ig, Proteintech), rabbit anti-Snail (3879, Cell Signaling Technology), mouse anti-Snail (sc-271977, Santa Cruz), rabbit anti-p66β (ab76925, Abcam), anti-biotin, HRP-linked (7075, Cell Signaling Technology), and rabbit anti-IgG (2729, Cell Signaling Technology), mouse anti-α-tubulin (66031-1-Ig, Proteintech), rabbit anti-Fibronectin1 (15613-1-AP, Proteintech), rabbit anti-E-cadherin (20874-1-AP, Proteintech), rabbit anti-Vimentin (10366-1-AP, Proteintech).

ASC antibody (1:200, Abcam, ab155970, UK) was used for immunofluorescence in P. gingivalis-treated BMDMs (MOI = 50), with or without pretreatment of OTTSP167 or Compound 50 for 2 h. Mouse acet-tubulin antibody (1:500, Proteintech, 66,200-1-Ig, China) and mouse anti-α-tubulin (1:500, Proteintech, 66,031-1-Ig, China) were used for staining the structure of microtubuli. The color reactions of images were captured on the confocal microscopy (Nikon A1, Japan).