Hrp conjugated goat anti rabbit igg secondary antibody

The cellular total protein were obtained by a protein extraction kit. Besides, the Bradford protein assay kit (BESTBIO) was used to determine the protein concentration. 30 μg or 50 μg of total cell extracts were analyzed by Western blotting. The proteins were loaded onto 15% SDS-PAGE gels and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). Membranes were incubated with 1 μg/ml Rabbit anti-ATG4a antibody (Polyclonal, ABGENT), 2 μg/ml mouse anti-SQSTM1/p62 antibody (Monoclonal, Abcam), 2 μg/ml rabbit anti-LC3 antibody (Cell Signaling Technology, Inc.), 2.5 μg/ml rabbit anti-GAPDH antibody (Monoclonal, Cell Signaling Technology, Inc.) or 2 μg/ml mouse anti-β-actin antibody (Monoclonal, Cell Signaling Technology, Inc.). After that, the membranes were incubated with 1 μg/ml HRP-conjugated goat anti-rabbit IgG secondary antibody or 1 μg/ml HRP-conjugated goat anti-mouse IgG secondary antibody (Proteintech Group, Inc.). Immunoreactive band analysis was performed by using the enhanced western bright ECL reagent (Advansta, United States). Densitometry analyses of western blots were performed with ImageJ software (version 1.46). Western blots were developed to be linear in the range used for densitometry. All results were expressed as a relative ratio to the β-actin or GAPHD. At least three or four independent western blot experiments were performed.

Protein extraction and western blotting were performed as previously described [16 (link)]. Briefly, HRGECs were collected and protein was extracted. Then, the protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Hereafter, the membranes were blocked with 5% BSA followed by incubation with rabbit anti-β-actin (1 : 1000), anti-TLR4 (1 : 1000), anti-MyD88 (1 : 1000), anti-NF-κB/p65 (1 : 1000), or anti-p-IκBα-Ser36 (1 : 2000) antibody overnight. Thereafter, horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG secondary antibody (Proteintech, Wuhan, China) (1 : 5000) was added for 2 h at room temperature. After washing with Tris-buffered saline with Tween 20 (TBST), the signals were detected using a LI-COR Odyssey Infrared Imaging System (Lincoln, NE, USA). All experiments were repeated at least three times.

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.).