Pkc activity assay kit

Immunoprecipitation (IP) was performed with cell lysates (~ 1.0 mg of protein) using the standard method in RIPA buffer without 0.1% SDS. Whole-cell lysates were pre-cleared using protein G-agarose beads (Invitrogen) for 1 h and precipitated overnight with an anti-PKCζ antibody at 4°C. The immunoprecipitates were washed five times with IP buffer and PKCζ was eluted using IgG elution buffer (Thermo Scientific, Wilmington, DE, USA). A PKC-specific kinase assay was performed using a PKC Activity Assay Kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions.

Previous studies have shown that 17β-estradiol can regulate metastasis and invasion through a rapid ERα36–dependent membrane–mediated pathway involving PLC and PKC.¹⁰ (link)^,16 (link)^,19 (link)^,23 (link)^,25–27 (link) To investigate if these signaling pathways are involved in the effect of E₂ on TNBC osteolysis, we evaluated the activity of both PLC and PKC after treatment with E₂ and E₂-BSA. MDA-MB-231 cells were cultured to confluence on six–well plates and treated with vehicle, 10^-8 M, or 10^-7 M E₂-BSA for 9 min. Cells were harvested immediately in 300 μL PLC lysis buffer or PKC lysis buffer according to the manufacturer’s protocol and assayed using the Amplex Red Phospholipase C Assay Kit (Thermo Fisher Scientific) or PKC Activity Assay Kit (Abcam), respectively. Cell layer lysates were assayed for total protein content using the Pierce BCA reagent kit (Thermo Fisher Scientific). PLC and PKC activities were normalized to total protein content per minute.