Anti tfeb antibody

Co-immunoprecipitation (CoIP) experiments were undertaken using a Dynabeads Protein G Immunoprecipitation kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, cell lysates were extracted from culture cells using Pierce IP Lysis Buffer (87787; Thermo Fisher Scientific) supplemented with protease inhibitor (Beyotime Biotechnology). After removing nonspecific binding by Dynabeads Protein G, the cell lysates were added with an appropriate amount of anti-TFEB antibody (catalog number A303-673A; Bethyl) or anti-RIP3 antibody (catalog number ab56164; Abcam), or control IgG and then incubated at 4 °C overnight. The Dynabeads-Ab-antigen complex was washed with washing buffer and eluted with RIPA buffer for western blotting.

BMDMs were cultured on coverslips in 24-well cell culture plates. After the appropriate infection or treatment, cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.25% Triton X-100 (Sigma-Aldrich) for 10 min. Cells were incubated with anti-TFEB antibody (1:400 diluted; Bethyl Laboratories, A303-673A) or anti-LAMP1 Ab (1:400 diluted; Santa Cruz Biotechnology, SC-19992) overnight at 4°C. Cells were washed with PBS to remove excess primary antibodies and then incubated with secondary anti-rabbit or anti-rat IgG-Alexa Fluor 488 Ab (1:400 diluted; Invitrogen, A11008 or A11006) for 1 h at RT. Nuclei were stained using Fluoromount-G™, with DAPI mounting medium (Thermo Fisher Scientific, 00-4959-52). Immunofluorescence images were acquired using a confocal laser-scanning microscope (Zeiss, LSM-900). Quantification of TFEB-nuclear translocation was performed by manual calculation and the degree of colocalization between Mtb-ERFP and LAMP-1 was analyzed using the JACoP plugin of the ImageJ software.