Western lightening chemiluminescence reagent plus

HaCaT human keratinocytes were lysed in PBS containing 1% Triton X-100 and 1 mM DTNB (Lee et al., 2005 (link)). The cell lysates and the concentrated conditioned buffer were diluted with 5× SDS sample buffer containing no reducing agent and incubated at room temperature for 5 min prior to loading onto the gels. Proteins were resolved by 7.5% SDS-PAGE, transferred to nitrocellulose membranes, and probed with the indicated mAbs. The binding of the mAbs was detected using HRP conjugated secondary antibodies, and visualized using Western Lightening Chemiluminescence Reagent Plus (Perkin-Elmer, Boston, MA).

Cells were lyzed in PBS containing 1% Triton X-100 and 1 mM DTNB. DTNB was added to the lysis buffer to prevent cleavage of disulfide linkages [30] (link). The protein concentration was determined by Bradford protein assay and equal amounts of proteins or equal proportions of cell lysates and conditioned buffers were analyzed by Western blot. Protein samples were diluted in 5× sample buffer containing no reducing agent and incubated at room temperature for 5 min. Proteins were resolved by 7.5% SDS-PAGE, transferred to nitrocellulose membranes, and then probed with the various mAbs. The binding of mAbs was detected using HRP conjugated secondary antibodies, and visualized using Western Lightening® Chemiluminescence Reagent Plus (Perkin-Elmer, Boston, MA).

Cytoplasmic extracts were obtained as previously reported 6 (link). The proteins (20 μg/lane) were run on 7.5, 10 or 15 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by semi-dry transfer to PVDF membrane (Invitrogen, Carlsbad, CA). Transferred PVDF blots were pretreated with 5 % non-fat dry milk in TBST containing 0.1 % Tween-20 and incubated with primary antibody (1:1000-3000) at 4 °C overnight. The membrane was then washed 3 times with TBST and incubated with horseradish peroxidase-conjugated secondary antibody (1:1000-3000) for 1 h at room temperature. For phosphorylated protein, transferred PVDF blots were pretreated with PVDF Blocking Reagent (TOYOBO, Osaka, Japan) for 1 h, and incubated with primary and then with secondary antibody (1:3000-6000) which were diluted with Can Get Signal® Immunoreaction Enhancer Solution (TOYOBO, Osaka, Japan) at room temperature for 1 h. After washing three times again, antibodies bound to protein blots were detected by using Western Lightening Chemiluminescence Reagent Plus (Perkin Elmer Life Science, Boston, MA, USA), visualized on LAS-3000 mini (FUJIFILM).