Polyclonal anti gfp antibody

Antibodies were from the following sources: monoclonal anti-PP2A_Cα/β (05-421), monoclonal anti-methyl PP2A_c (2A10) and monoclonal anti-demethylated PP2A_C (4B7) antibodies were purchased from Millipore/Upstate Biotechnologies, UK; polyclonal anti-Gα_t1 (K20), monoclonal anti-LCMT-1 (4A4), polyclonal anti-PP2A_C (FL-309) and monoclonal anti-PME-1 (B12) antibodies were purchased from Santa Cruz Biotechnology; polyclonal anti-cardiac troponin I (cTnI), monoclonal anti-PKB (2H10), rabbit monoclonal anti-PKB (11E7) and phosphorylated PKB (ser473) were obtained from Cell Signaling Technology. Polyclonal anti-GFP antibody was purchased from Clontech; anti-B55α (PPP2R2A) polyclonal antibody was purchased from Merck Calbiochem; HRP-conjugated donkey anti-rabbit and sheep anti-mouse secondary antibodies were purchased from GE Healthcare, UK. G_iPCR agonists N⁶-cyclopentyladenosine (CPA), carbachol (CCH) and CGP42112 (CGP) were purchased from Sigma-Aldrich; LY294002 was purchased from Merck Calbiochem. Recombinant adenovirus coexpressing enhanced green fluorescent protein (EGFP) and the Gα subunit of transducin (Gα_t1) was a kind gift from Professor Thomas Wieland [27] (link), University of Heidelberg, Germany.

For constructing the kinA^(1–894)-GFP strain, we made the kinA^(1–894)-GFP-AfpyrG fragment by inserting the GFP-AfpyrG fragment into the kinA^K895* mutation site right before the stop codon. The following four oligos were used to make the kinA^(1–894)-GFP-AfpyrG construct: K5U, K895.R (5′-GCA​CCA​GCT​CCA​GCG​ATT​CGG​GAG​CCA​G-3′), K895.F (5′-AAT​CGC​TGG​AGC​TGG​TGC​AGG​CG-3′), and BW6. Specifically, K5U and K895.R were used to amplify a ∼0.9-kb fragment of the 5′ coding region, and K895.F and BW6 were used to amplify a ∼3.6-kb fragment using genomic DNA from the RQ197 strain (containing kinA-GFP) as template. We then used two oligos, K5U and BW6, for a fusion PCR to fuse the two fragments to generate the ∼4.5-kb kinA^(1–894)-GFP-AfpyrG fragment that we used to transform the XY42 strain and the RQ54 strain. The transformants were screened by microscopically observing the GFP signals and further confirmed by a Western blotting analysis with a polyclonal anti-GFP antibody from Clontech. In addition, we also performed a diagnostic PCR to verify the correct integration using oligos K5UTR (5′-GAA​CGA​CCT​CAC​AGA​CTC​A-3′) and GFP-5R (5′-CAG​TGA​AAA​GTT​CTT​CTC​CTT​TAC​T-3′).