Nitrocellulose pvdf membranes

Lysates generated from BMDMs, were separated by 15 % SDS-PAGE and transferred onto nitrocellulose PVDF membranes (Millipore). Blots were immersed in 5 % milk powder in TBS/tween and incubated overnight at 4 °C with a rat monoclonal antibody for IL-1β (RnD Systems AF-501). Membranes were then washed and incubated with rabbit anti-rat secondary antibody (Abcam). Proteins were imaged using ECL Plus chemi luminescent substrate (ThermoFisher Scientific) and exposed using the ChemiDoc XRS imaging system (BioRad Laboratories).

Supernatants from BMDMs that had been treated with LLOME for the indicated time-points were diluted in MES reaction buffer and incubated at 37 °C for 15 mins with 10 μM biotin-PEG-LVG-DMK. The reaction was terminated by addition of 5X Laemmli buffer and denaturation at 95 °C. Samples were resolved by 15 % SDS-PAGE and transferred onto nitrocellulose PVDF membranes (Millipore), blots were subsequently blocked in 5 % BSA in TBS/tween overnight at 4 °C. The membrane was then incubated with Streptavidin-horse radish peroxidase (Cell Signaling) diluted TBS/Tween, with 5 % BSA for 0.5 h at room temperature. Proteins were imaged using ECL Plus chemiluminescent substrate (ThermoFisher Scientific) and exposed using the ChemiDoc XRS imaging system (BioRad Laboratories).

To further verify the variation in the DEPs identified by the proteomic approaches, HSPA1B was selected for western blot analysis. The CPIV3- and mock-infected cells were collected at 24 and 48 hpi. Equivalent amounts of cell lysate from each sample were collected. After measuring the protein concentrations, equivalent amounts of cellular proteins were separated by SDS-PAGE and transferred onto nitrocellulose PVDF membranes (Millipore, USA). The membranes were incubated overnight at 4 °C with primary rabbit polyclonal antibodies of anti-HSPA1B (Biyotime, Shanghai, China). Then the membranes were further incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (BIOSS, Beijing, China). The protein bands were detected using the ECL Detection Kit (Vazyme, Nanjing, China). β-actin protein was used as an internal control.