Mouse anti his

Western blot analysis was done as described previously [48 (link)], using a 1:1,000 dilution of polyclonal rabbit anti-PKAR3, anti-TbPKAR1 [7 (link)], anti-TbPKAC1/C2 [12 (link)] and anti-TbSAXO [25 (link)]. Rabbit antibodies against TbPKAC3 were used at 1:250 dilution [12 (link)]. For western blot detection of proteins recombinantly expressed in L. tarentolae, mouse anti-His (Bio-Rad) and mouse anti-Strep (Qiagen) were used. The secondary antibodies IRDye680LT goat anti-rabbit (1:50,000; LICOR) and IRDye800CW goat anti-mouse (1:10,000; LICOR) were used for detection with the Odyssey CLx imaging system (LICOR). Rabbit antiserum against L. donovani HSP83 [28 (link)] or β-tubulin (Cell Signalling) were used as protein loading markers.

The antigenic preparation was electrophoresed in sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) using 4% stacking gel and 12% separating gel cast in Mini-PROTEAN Cell (Bio-Rad, Hercules, California, USA). The SDS-PAGE-separated components in the separating gels were electroblotted onto nitrocellulose membrane (NC) and the unoccupied sites on the blotted NC were blocked with 5% skim milk in Tris-buffered saline containing 0.01% Tween-20 (TBS-T) for 1 h. Excess blocking reagent was discarded; the NC was washed three times with the TBS-T and placed in a solution of mouse mAb to HuscFv34, mouse anti-His (Bio-Rad), or mouse anti-SUMO (Genscript) at RT for 1 h. The NC was washed again with the TBS-T and incubated with goat-anti-mouse Ig-alkaline phosphatase (AP) conjugate (SouthernBiotech, Birmingham, AL, USA) for 1 h. After washing with TBS-T, BCIP/NBT substrate (KPL, SeraCare, Millford, MA, USA) was used to reveal the antigen-antibody reactive bands. The NC was washed with distilled water and air-dried.