hek 293 lysis buffer  (Thermo Fisher)

Journal: iScience

Article Title: PKCμ promotes keratinocyte cell migration through Cx43 phosphorylation-mediated suppression of intercellular communication

doi: 10.1016/j.isci.2024.109033

Figure Lengend Snippet: PKCμ interacts with Cx43 and phosphorylates Cx43 at S368 (A) In vitro pull-down assay with HA-PKCμ attached to HA antibody conjugated agarose beads were incubated with either GST or GST-Cx43 and subsequently washed to remove unbound material. Pulled down material was probed by western blotting for GST and HA. (B) In vitro kinase assay with HA-PKCμ immunoprecipitated from untreated or PMA treated transfected HEK 293T cells incubated with bacterially expressed GST-Cx43 in the presence or absence of ATP (200 μM). Reactions were probed by western blotting for pCx43-S368, HA, and GST. (C) In vitro kinase assay as described in (B) with wild-type (WT) or catalytically inactive (K612W) HA-PKCμ and GST-Cx43. Reactions were probed by western blotting for pCx43-S368, HA, and GST.

Article Snippet: In vitro kinase assay was carried out with 4 μg GST or GST-Cx43 and agarose-conjugated HA-PKCμ protein isolated from HEK 293T cells combined with kinase buffer from Cell Signaling Technology in the absence or presence of ATP and incubated for 30 minutes at 30 ° C. Reactions were terminated by addition of 1X Laemmli buffer.

Techniques: In Vitro, Pull Down Assay, Incubation, Western Blot, Kinase Assay, Immunoprecipitation, Transfection