Hrp conjugated goat anti mouse igg

Western blot was performed as previously described with minor modifications (Zhang et al., 2019 (link)). Briefly, harvested cells were lysed in RIPA buffer, and protein concentrations were determined via BCA assay follow the manufacturer's instructions. Total protein (50 μg) was resolved (SDS‐PAGE, 8%–15% gradient gels) and transferred to PVDF membranes. Immunoblotting was carried out using primary antibodies and horseradish peroxidase‐conjugated secondary antibodies. The blots were visualized using a Tanon 5200 automatic chemiluminescence image analysis system (Tanon, Shanghai, China).
The following antibodies were used: anti‐ERK (1:1000, cat no. 4695S, CST), anti‐p‐ERK (1:500, cat no. 9101S, CST), anti‐PI3K (1:1000, cat no. 4249S, CST), anti‐p‐PI3K (1:500, cat no. 4228S, CST), anti‐GAPDH (1:2000, cat no. UM4002, UtiBody), HRP‐conjugated goat anti‐rabbit IgG (1:3000, cat no. bs‐0295G‐HRP, Bioss) and HRP‐conjugated goat anti‐mouse IgG (1:3000, cat no. bs‐0296G‐HRP, Bioss).

Frozen testis sections and cultured cells were fixed with 4% paraformaldehyde for 10 min and washed twice with PBS. Later, they were blocked with 5% goat serum (Gibco) for at least 1 h and incubated with antibodies for 2 h (1:200 dilution in PBS). After being washed with PBST containing 0.1% Tween 20 (Beyotime), they were incubated with HRP-conjugated goat anti-rabbit IgG (Bioss) or HRP-conjugated goat anti-mouse IgG (Bioss) (1:2000 dilution in PBS) for 2 h. Antibody signals were colored using the TSA Plus Fluorescence Systems (PerkinElmer Life Science, Waltham, MA, USA). Nucleus was stained by propidium iodide. Photographs were imaged by a Zeiss SML800 laser scanning confocal microscope (Zeiss, Jena, Germany) or a Leica TCS SP5 laser scanning confocal microscope (Leica).

HEK293T cells were cotransfected with the pCDH-CMV-NS4A-Flag plasmids and three other plasmids (pGag/Pol, pRev, pVSVG) using TurboFect (Thermo Fisher Scientific, USA). After 16 h, the medium was replaced with Advanced DMEM. The culture supernatants containing lentivirus were collected after 48 h. PK-15 cells were infected with lentiviral products expressing NS4A and then selected with 6 µg/mL puromycin (Thermo, USA) for 10 days. To confirm that PK-15 cells expressed NS4A, green fluorescence in PK-15 cells was confirmed to be visible under an inverted fluorescence microscope (Nikon, Japan). Then, NS4A gene expression was analysed by western blotting in PK-15 cells expressing NS4A. In brief, PK-15 cells expressing NS4A were harvested and lysed. Protein samples were separated by 12% SDS‒PAGE. According to the size of target proteins and the indication of protein marker, the unused parts of the separating gel and stacking gel were cut off. The proteins were transferred onto PVDF membranes (Millipore, USA). The membranes were incubated with an anti-β-actin mouse mAb (Tianjin Sungene Biotech, KM9001, China) or anti-Flag mouse mAb (CWBIO, China) at 4 °C overnight and then incubated with HRP-conjugated goat anti-mouse IgG (1:5000) (Bioss, China) for 2 h at room temperature. Then, the signal was detected using an automatic chemiluminescence image analysis system (Tanon, 5200, China).