Anti ppkcα

The protein samples were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresed proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany), then blocked with 5% bovine serum albumin (BSA, Gbcbio, China). Membranes were probed with anti-p-MLC (#3674T, 1:1000, CST, USA), anti-MLC (#3672S, 1:1000, CST, USA), anti-p-pkcα (#sc-377565, 1:500, SantaCruz, USA), anti-pkcα (#2056S, 1:1000, CST, USA), anti-MLCK (#BM4290, 1:400, BOSTER, China), anti-Calmodulin (#bs-3666R, 1:500, Bioss, China), and anti-actin (#66009-1-Ig, 1:6000, Proteintech, China), followed by horseradish-peroxi-dast-conjugated goat anti-rabbit IgG secondary antibody (#SA00001-2, 1:6000, Proteintech, China) or horseradish-peroxi-dast-conjugated goat anti-mouse IgG secondary antibody (#SA00001-1, 1:6000, Proteintech, China) for 1 h. The blots were detected using an ECL chemiluminescence substrates kit (#WBKLS0100, Millirore, USA).

iMoDCs, plated at 1x10⁵/well in 6-well plates in a final volume of 1 ml, were serum starved for 4 hours and then stimulated in IMDM containing 1% FCS (BioWhittaker) and 20 μg/ml Bet v 1, Api g 1, or a control stimulus for 5, 10, 20, and 30 minutes or left untreated. The control stimulus solution contained 50 ng/ml TNF-α (Strathmann, Hamburg, Germany), 10 ng/ml IL-1β, and 500 U/ml GM-CSF (both PeproTech, Rocky Hill, NJ, USA) and is referred to in the text as maturation-inducing factors (MFs). Cells were lysed in 90 μl Laemmli buffer and the lysates heated for 5 min at 95°C. Protein extracts were separated on 10% SDS gels and transferred to a nitrocellulose membrane (GE Healthcare, Maidstone, UK). Blocking was carried out for 1 hour at room temperature with PBS containing 0.1% Tween 20 (PBST) and 5% skim milk. Primary antibodies (anti-PKCα, anti-pPKCα, anti-Erk1/2 from Santa Cruz Biotechnology, Santa Cruz, CA, USA; anti-pErk1/2, anti-pp38, and anti-IκBα from Cell Signaling Technology, Beverly, MA, USA) were diluted in PBST containing 5% BSA and incubated overnight at 4°C. Bound antibodies were visualized by anti-IgG antibodies conjugated with peroxidase (Sigma-Aldrich) and subsequent chemiluminescence detection (LumiGLO, Cell Signaling Technology).