Anti flag rabbit mab

Western blots were performed as described previously 24 (link). The following primary antibodies were used: mouse anti-P2X2 monoclonal antibody (mAb) (1:500; Youke Biotech); rabbit anti-Flag mAb (1:1,000; Sigma-Aldrich, F7425); mouse anti-GAPDH mAb (1:5,000; Good Here, AB-P-R001); The polyvinylidene fluoride (PVDF) membranes were washed and incubated for 1 h at room temperature with the corresponding secondary antibodies: horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5,000; ZSGB-Bio); HRP-conjugated goat anti-mouse IgG (1:5,000; ZSGB-Bio). Peroxidase activity was detected with SuperSignal WestPico chemiluminescent substrate (Pierce Biotechnology) and visualized and digitized with BIO-RAD Gel Doc XR imaging system (BIO-RAD, Germany). Protein levels, quantified by computer analysis as the ratio between each immunoreactive band and the levels of GAPDH, were expressed as a fold change of vehicle-treated control.

Polyclonal antibody to B. cenocepacia Hcp was raised in rats at the University of Sheffield Biological Services unit using N-terminal His-tagged Hcp which was purified following its overproduction in E. coli as described below. Rabbit anti-FLAG mAb and anti-VSVg pAb were obtained from Sigma-Aldrich (cat. no. F7425 and V4888, respectively), whereas rabbit anti-HA mAb was purchased from Cell Signaling Technology (cat. no. C29F4). HRP-conjugated mouse anti-MBP polyclonal antibodies were obtained from New England Biolabs (cat. no. E8038S). The mouse mAb to the β subunit of E. coli RNA polymerase was obtained from Neoclone (cat. no. W0023). Proteins harbouring a polyhistidine tag were detected using the HisProbe-HRP conjugate (Thermo Fisher, cat. no. 15165) or mouse anti-KLH-conjugated His-tag peptide (BioLegend, 652501). HRP-linked goat-derived (secondary) antibodies that cross-react to rat and rabbit IgG were obtained from SouthernBiotech (cat. no. 3050–05) and Vector Laboratories Ltd (cat. no. PI-1000), respectively. HRP-conjugated rabbit anti-mouse IgG secondary antibody were purchased from Thermo Fisher (cat. no. 31450).

HeLa cells were seeded on sterile glass coverslips in 6-well plates then transfected with pCAGGS-PCSK9-Flag and/or pCAGGS-CD163-HA. Twenty-four hours post-transfection (hpt), the cells were washed with PBS twice, fixed with 550 uL of cold methyl alcohol for 10 min, and blocked with 5% bovine serum albumin (Shenggong, Shanghai, China) for 30 min. The transfected cells were washed with PBS three times, incubated with mouse anti-HA Mab (Sigma, Shanghai, China, 1:2000) and rabbit anti-Flag Mab (Sigma, Shanghai, China, 1:2000) for 1 h at 37 °C, and washed three times with PBS. The cells were then incubated at 37 °C for 1 h with donkey anti-mouse IgG (H + L) antibody conjugated with Alexa Fluor 596 (Life Technologies, Shanghai, China, 1:800) and goat anti-rabbit IgG (H + L) antibody labeled with Alexa Fluor 488 (Life Technologies, Shanghai, China, 1:800). The cells were counterstained with 5 μg/mL of 4′, 6′-diamidino-2-phenylindole (DAPI, Beyotime, Shanghai, China) for 10 min. Images were taken using a Zeiss confocal system (Zeiss, Germany).