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A549 cells were grown in 6-well plates to 90% confluence and then infected with AH05 (H5N1) virus (MOI = 5) for 1 h on ice at 4°C. The infected cells were washed five times with ice-cold PBS (pH 7.2) or PBS-HCl (pH 1.3). Afterwards, the cells were lysed with SDS-PAGE loading buffer (Solarbio, Beijing, China) and then subjected to Western blotting with a rabbit anti-NP pAb.
A549 cells in 6-well plates were treated with siRNA targeting FFAR2 or AP2B1 or with scrambled siRNA (30 nM) for 48 h and then infected with AH05 (H5N1) virus (MOI = 5) on ice at 4°C for 1 h, followed by a culture temperature shift to 37°C for 30 min to allow for internalization. The cells were then washed five times with ice-cold PBS-HCl (pH 1.3) to remove the attached but not-yet-internalized virions and then subjected to Western blotting with a rabbit anti-NP pAb.
A549 cells were grown in 6-well plates to 90% confluence and then treated with DMSO, 4-CMTB (100 μM), or Cmp58 (10 μM) for 3 h at 37°C prior to infection with AH05 (H5N1) virus (MOI = 5) at 37°C. The cells were washed five times with ice-cold PBS-HCl (pH 1.3) at the indicated time points p.i. to remove the attached but not-yet-internalized virions and then subjected to Western blotting with a rabbit anti-NP pAb.

The cells were washed with ice-cold PBS three times and lysed with RIPA lysis buffer (Sangon Biotech, China) with a protease inhibitors cocktail (Roche, China). The total protein content in the supernatant was measured by the BCA protein assay kit (Sangon Biotech, China) according to the manufacturer’s instruction. The samples were boiled with SDS-PAGE loading buffer (Solarbio, China). Twenty micrograms of protein was added to each lane, separated in 10% SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA) in a Trans-Blot apparatus (Tanon, China). 5% BSA diluted in TBS-T (0.05% Tween 20 in TBS) was used to block the membranes, and the primary antibodies with optimized concentrations, including AR (1:1000, ab108341, Abcam, USA), Actin (1:1000, D191048, Sangon Biotech, China), PSA (1:1000, 10679, Proteintech, China), were added to the membranes overnight at 4 °C. The membranes were incubated with the corresponding secondary antibodies conjugated with HRP (1:10000, D110011, Sangon Biotech, China) for 1 h at room temperature, and the bands were visualized by an enhanced chemiluminescence detection system.

The total proteins were extracted from cells with radioimmunoprecipitation assay (RIPA) lysis buffer mixed with PMSF, protein inhibitors, and phosphatase inhibitors (Beyotime, China) and denatured by SDS‐PAGE loading buffer (Solarbio, China). An equal quantity of protein was separated using smartPAGE™ precast protein gel plus (4%–20%, Bis‐Tris) (Smart‐Lifesciences, China) in MOPS Running Buffer (Smart‐Lifesciences, China) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA) in NcmBlot Rapid Transfer Buffer (NCM Biotech, China). The membranes were blocked with 5% nonfat dry milk (Beyotime, China) for 1.5 h and then incubated with primary antibodies: METTL14 polyclonal antibody (26158‐1‐AP, 1:1000, Proteintech, China), p21 polyclonal antibody (10355‐1‐AP, 1:1000, Proteintech, China), p53 polyclonal antibody (10442‐1‐AP, 1:1000, Proteintech, China), FTO polyclonal antibody (27226‐1‐AP, 1:1000, Proteintech, China), collagen type I polyclonal antibody (14695‐1‐AP, 1:1000, Proteintech, China), ERRFI1 polyclonal antibody (11630‐1‐AP, 1:1000, Proteintech, China), and GAPDH polyclonal antibody (10494‐1‐AP, 1:20000, Proteintech, China). Proteins were then incubated by anti‐rabbit IgG, HRP‐linked antibody #7074 (CST, USA) for 1.5 h and detected by an enhanced chemiluminescence system (ECL) reagent (Biosharp, China).