Rabbit anti lc3 primary antibody

The inhibitory effects of miR-20a on autophagy were evaluated by counting LC3 puncta in RAW264.7 cells after BCG infection. The RAW264.7 cells were transfected with miR-20a control, miR-20a mimic, miR-20a inhibitor, ATG7 siRNA, ATG7 siRNA plus rapamycin, ATG7 siRNA plus miR-20a mimic, and then treated with BCG at an MOI of 10 for 24 h. The RAW264.7 cells were fixed with 4% paraformaldehyde. The RAW264.7 cells were blocked with 3% bovine serum albumin (BSA) and incubated with 10 μg/ml Rabbit anti-LC3 primary antibody (Cell Signaling Technology, Inc.) and then 2 μg/ml Alexa Fluor 488–conjugated goat anti-rabbit IgG (Abcam) before mounting. The images of cells were visualized and acquired using an Olympus DSU spinning disk confocal microscope under a 100 × objective oil lens. The number of endogenous LC3 punctate dots was counted by using ImageJ Software version 1.46. At least 15 cells per experimental group were counted and each condition was assayed in triplicate. The LC3-II protein levels were evaluated by Western blot using Rabbit anti-LC3 primary antibody (Cell Signaling Technology, Inc.) and HRP-conjugated Goat anti-Rabbit IgG secondary antibody (Proteintech Group, Inc.).

The RAW264.7 cells were transfected with miR-144-3p control, miR-144-3p mimic or miR-144-3p inhibitor for 24 h, or treated with 50 μg/ml rapamycin (MedChemExpress) in 0.1% DMSO. After that, the RAW264.7 cells were infected with BCG for 24 h. The cells were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100 (Sigma) for 10 min, and incubated with 10 μg/ml rabbit anti-LC3 primary antibody (Cell Signaling Technology, Inc.) at 4℃ for overnight. The cells were then incubated with 2 μg/ml Alexa Fluor 488–conjugated goat anti-rabbit IgG (Abcam) at room temperature for 1 h. Nuclei were stained with Prolong Gold anti-fade reagent with 4’-6-diamidino-2-phenylindole (DAPI) (P36931, Invitrogen, Grand Island, NY, USA) for 1 min. The fluorescence images were acquired using an Olympus DSU spinning disk confocal microscope under a 100 × objective oil lens. To quantify autophagy, endogenous LC3 punctate dots were determined using ImageJ software (version 1.46). At least 15 cells were counted per condition in each experimental group and assays were repeated in triplicate for each condition.