Fig. 6.

Endogenous expression of calmodulin-dependent protein kinase II (CaMKII) and the Ca²⁺-independent protein phosphatases PP1 and PP2A in nontransfected HEK-293 cells. A: immunocytochemical detection of CaMKII (top left) and PP2A (top middle) protein expression was respectively performed using a primary rabbit polyclonal antibody against CaMKII from Santa Cruz Biotechnology (Dallas, TX; sc-9035) and a rabbit monoclonal antibody against the catalytic subunit of PP2A from Cell Signaling Technology (Danvers, MA; 52F8). Detection of both enzymes was carried out by incubating cells with a secondary anti-rabbit IgG Alexa Fluor 546 (Thermo Fisher Scientific, Waltham, MA; A-11010). Cells serving as negative controls were incubated with the secondary antibody only (2nd Only; top right). Immunocytochemical studies were also carried out to determine the endogenous expression of the catalytic subunits of PP1α (bottom left top), PP1β (bottom right top), and PP1γ (bottom left lower) in cultured HEK-293 cells. All antibodies were goat antibodies from Santa Cruz Biotechnology (PP1α: sc-6105; PP1β: sc-6107; PP1γ: sc-6109). All cells were incubated with the goat anti-rabbit IgG Alexa Fluor 488 (Thermo Fisher Scientific; A-11008) secondary antibody. Negative controls were labeled with secondary antibody only (2nd Only; bottom right lower). A ×60 oil immersion objective was used for detection of all enzymes. For all images in A, nuclei were stained with DAPI (Abcam, Cambridge, UK). B: expression of CaMKII isoforms at the mRNA level was determined using specific primers designed against each of the 4 CaMKII isoforms (see Table 1). For each set of primers pairs, a nontemplate control (NTC) was performed; however, for the sake of space, this figure shows a single representative NTC. PCR amplified products were resolved on a 2% agarose gel. Single bands for the CaMKIIβ and -δ isoforms were detected in HEK-293 cells, which fall within the expected amplicon size (396 bp for both) based on primer design. Multiple bands were detected in HEK-293 cells for CaMKIIα and -γ, which is most likely the result of alternative splicing. Predicted amplicon size for CaMKIIα: 452 bp; CaMKIIγ: 413 bp; CaMKIIδ: 396 bp. C: Western blot analysis of nontransfected HEK-293 cells demonstrated the expression of several isoforms of CaMKII, all three known catalytic PP1 isoforms, and two isoforms of the catalytic subunit of PP2A. Proteins of interest and ladder appear in red while the housekeeping proteins GAPDH and β-actin appear in green. 20 μg of lysate protein were loaded in each lane.