MDMs were infected at a MOI of 50 for 30 min for studies of ROS production. Supernatants were removed post treatment and the cells were washed twice with PBS (Fisher). The cells were lysed in lysis buffer (HEPES, MgCl, EGTA, KCL, NP-40) with protease inhibitor (Roche Applied Science, 10-519-978-001). Then, 30 ug of protein was separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were immunoblotted for calreticulin (Enzo Life Sciences, ADI-SPA-600), phospo-p40phox (Cell Signaling, 4311), total p40phox (Abcam, ab137691), phospho-p47phox (donated by Jamel El-Benna), total p47phox (Life Technologies, A16636), p67phox (Santa Cruz, SC-15342), Rac2 (Abcam, ab2244), gp91phox (Santa Cruz, SC-5827), p22phox (Santa Cruz, SC-20751),) p-PKC α/β II (Cell Signaling, 9375P), p-PKC δ (Cell Signaling, 9374P), and p-PKC ζ/λ (Cell Signaling, 9378P). Protein bands were detected with HRP-conjugated secondary antibodies and visualized using ECL reagents (Life Sciences, RPN2106). Membrane and cytosolic fractionations were prepared via manufacturer kit instructions (Thermo Fisher, #78840).
P pkc α β 2
Manufactured by Cell Signaling Technology
The ),) p-PKC α/β II is a lab equipment product manufactured by Cell Signaling Technology. It is an antibody that detects endogenous levels of PKC α or PKC β II protein when phosphorylated at specific sites.
Automatically generated - may contain errors
Lab products found in correlation
P pkcδ, by Cell Signaling Technology (2 mentions)
Gp91phox, by Santa Cruz Biotechnology (1 mentions)
P22phox, by Santa Cruz Biotechnology (1 mentions)
P67phox, by Santa Cruz Biotechnology (1 mentions)
P pkc ζ λ, by Cell Signaling Technology (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Anti bip, by Cell Signaling Technology (1 mentions)
Cyt c, by Cell Signaling Technology (1 mentions)
Hsp70, by Cell Signaling Technology (1 mentions)
P p53 s15, by Cell Signaling Technology (1 mentions)
Hsp60, by Cell Signaling Technology (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
2 protocols using p pkc α β 2
1
Measuring Oxidative Stress Signaling
2
Euphol-induced Protein Expression Analysis
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To evaluate the expression of altered proteins following euphol treatment, the cells were plated onto a six-well plate at a density of 1 × 10 6 cells/well, allowed to adhere at least 24 h, and were serum-starved in DMEM (0.5% FBS). The cells were exposed at IC 50 concentration values of euphol for an additional period of 6, 24, 48 and 72 h in DMEM (0.5% FBS). After the cells were prepared and aliquots of 20 μg of total protein were separated as previously described for western-blot analysis [27] (link). Antibodies included anti-Bip, Rip, p-27, DR5, SOD2, CytC, pP53 (S15), HSP60, HSP70, FADD and β-tubulin, all antibodies were diluted at 1:1000, (Cell Signalling Technology). In addition to PKC profile analysis, antibodies included total PKCs (PKCα, PKCδ and PKCζ), and phosphorylated PKCs; p-PKC PKDμ (S916), p-PKC PKDμ (S744), p-PKCα/βII, p-PKCpanβII, p-PKCδ, p-PKCδ/θ, p-PKCθ and PKCζ/λ, all antibodies were diluted at 1:1000 and purchased from Cell Signalling Technology.
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