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 ζ λ
Manufactured by Cell Signaling Technology
P-PKC ζ/λ is a primary antibody that recognizes the phosphorylated forms of the protein kinase C (PKC) isoforms ζ and λ. PKC isoforms are important signaling molecules involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
P pkcδ, by Cell Signaling Technology (1 mentions)
P pkc α β 2, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Gp91phox, by Santa Cruz Biotechnology (1 mentions)
P22phox, by Santa Cruz Biotechnology (1 mentions)
P67phox, by Santa Cruz Biotechnology (1 mentions)
Acetyl coa carboxylase, by Cell Signaling Technology (1 mentions)
P acc, by Cell Signaling Technology (1 mentions)
Ampkα2, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor, by Cell Signaling Technology (1 mentions)
Bca assay, by Beyotime (1 mentions)
Protease inhibitor, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
2 protocols using p pkc ζ λ
1
Measuring Oxidative Stress Signaling
2
Protein Extraction and Western Blotting
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Samples were ground and lysed in RIPA buffer containing 1% protease inhibitor and 1% phosphatase inhibitor (Cell Signaling Technology, Beverly, MA, USA) and centrifuged for 12 min at 12 000 g , 4 °C to remove debris. A BCA assay was used to determine protein concentrations (Beyotime, Shanghai, China). About 50 μg total protein for each sample was separated by SDS/PAGE and transferred to PVDF membrane. Membranes were incubated with the following primary antibodies: AMPKα2 (1 : 1000; Cell Signaling Technology), phospho (p)‐AMPKα2 (Thr172, 1 : 1000; Cell Signaling Technology), acetyl‐CoA carboxylase (ACC) (1 : 1000; Cell Signaling Technology), p‐ACC (Ser79, 1 : 1000; Cell Signaling Technology), mechanistic target of rapamycin (mTOR) (1 : 1000; Cell Signaling Technology), p‐mTOR (Ser248, 1 : 1000; Cell Signaling Technology), protein kinase C ζ/λ (PKCζ/λ) (1 : 1000; Cell Signaling Technology), p‐PKCζ/λ (Thr410/403, 1 : 1000; Cell Signaling Technology), and β‐actin (1 : 1000; Abcam). Signals were visualized by enhanced ECL substrate (Beyotime).
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