Journal: The Journal of biological chemistry

Article Title: The N terminus of the cardiac L-type Ca(2+) channel alpha(1C) subunit. The initial segment is ubiquitous and crucial for protein kinase C modulation, but is not directly phosphorylated.

doi: 10.1074/jbc.274.44.31145

Figure Lengend Snippet: FIG. 1. Detecting the Ca21 channel a1C subunit isoforms in Xenopus oocytes and in tissues with Card-N, Card-I, and Card-C antibodies. A, Card-N and Card-C immunoprecipitate [35S]methionine/cysteine-labeled a1C from oocytes expressing the WT a1C (lanes 2 and 4) but not from uninjected oocytes of the same donor (lanes 3 and 5). Card-N fails to immunoprecipitate a1CDN2–46 (lane 1). B, Card-C and Card-I immunoprecipitate a1CWT (lanes 2 and 5) and a1CDN2–46 (lanes 3 and 6) from oocytes of one donor injected with the corresponding RNAs, but not from uninjected oocytes (lanes 1 and 4). In panels A and B, a1C was coexpressed with a2/d. In each lane, immunoprecipitates from 5 oocytes were loaded. C, Western blot of rat ventricular membranes with the Card-N antibody in the absence (lane 2) or presence (lane 1) of the GST-fusion protein N1–46(S44A). 10 ml of the antibody (dilution 1:1000) were incubated overnight at 4 °C with 80 mg of N1–46(S44A), then additional 80 mg were added, and the antibody/GST fusion protein mixture (lane 1), or 10 ml of the antibody without N1–46(S44A) (lane 2), were incubated with the nitrocellulose membranes for 2 h at room temperature. D, detecting the Ca21 channel a1C subunit isoforms in ventricle, brain, and liver by immunoblots with Card-N, Card-I, and Card-C.

Article Snippet: To eliminate anti-GST antibodies, the IgG fraction was incubated with GST beads (Affi-Gel 10, Bio-Rad) overnight at 4 °C.

Techniques: Labeling, Expressing, Injection, Western Blot, Incubation

Journal:

Article Title: Functional Interactions between Herpesvirus Oncoprotein MEQ and Cell Cycle Regulator CDK2

doi:

Figure Lengend Snippet: The serine 42 residue is the primary site of phosphorylation by CDKs. (A) An array of bacterially expressed truncation or point mutants of MEQ protein was purified with Talon (Clontech). (B) MEQ-bZIP (S42G) is not appreciatively phosphorylated by CDK1-cyclin B. In vitro kinase assays were performed on MEQ bZIP and mutant proteins with CDK1-cyclin B complex in the presence of [γ-32P]ATP at 37°C for 30 min. The samples were resolved by SDS-PAGE and exposed to an X-ray film for 3 min. The same amount of MEQ mutant protein was loaded on a separate SDS-PAGE gel, and the Western blot was detected with rabbit anti-MEQ polyclonal antibodies (1:4,000 dilution). (C) MEQ-bZIP and MEQ-bZIP (S42G) mutant proteins were also phosphorylated by other serine/threonine kinases, including PKA, PKC, and MAPK, in vitro, and the gels were exposed to X-ray films at different intervals (5 min for PKA and PKC and 15 min for MAPK). (D) MEQ is a phosphoprotein in vivo. MEQ, in the context of pTM1 vector with an EE epitope tag, was transfected into CV1 cells with recombinant vaccinia virus. At 24 h later, the cells were labeled with [γ-32P]ATP and MEQ protein was immunoprecipitated with EE epitope MAb-conjugated Affi-Gel 10 beads. Purified protein was resolved by SDS-PAGE, and the blot was probed against MEQ polyclonal antibodies. CV-1 cells were also transfected with PTM1 vector alone as a negative control.

Article Snippet: EE epitope-tagged MEQ and cyclin A, B, and E proteins were expressed in CV-1 cells with the vaccinia virus-T7 polymerase expression system and immunoprecipitated with 20 μl of EE epitope MAb-conjugated Affi-Gel 10 beads (Bio-Rad) as described previously ( 24 ).

Techniques: Purification, In Vitro, Mutagenesis, SDS Page, Western Blot, In Vivo, Plasmid Preparation, Transfection, Recombinant, Labeling, Immunoprecipitation, Negative Control