Anti ha antibody

Immunofluorescence analysis was carried out as described earlier (53 (link)). Briefly, 5–7 × 10⁶ logarithmically growing promastigotes were collected by centrifugation, washed with 1XPBS, fixed in 2% paraformaldehyde, and cells spread on poly-lysine coated coverslips before permeabilization with 0.1% TritonX-100. The permeabilized cells were blocked with 5% chicken serum, incubated with anti-HA antibody (1:100 dil; Cell Signaling Technology; Catalog no: C29F4), washed, incubated with Texas Red-labelled anti-rabbit secondary antibody (1:100 dil; Jackson ImmunoResearch Laboratories), washed, and mounted in DAPI-containing anti-fade medium (Vectashield from Vector laboratories). Cells were observed by excitation at 594 nm and monitoring emission at 615 nm, using a 100× (in oil) objective under a Leica TCS SP8 confocal microscope, and Z-stack images were collected and analyzed with the LAS X software.

After trichloroacetic acid treatment, cells were lysed in the sodium dodecyl sulfate sample buffer as described previously [34 (link)] and subjected to western blotting. Proteins on the blot were detected by anti-HA antibody (rabbit, 1:2000, Cell Signaling, 3724) or anti-alpha-tubulin antibody (mouse, 1:8000, Sigma, T6199), followed by the incubation with the secondary antibodies Goat anti-Rabbit IgG (H + L) HRP Conjugate (1:8000, TransGen Biotech, HS101-01) and Goat anti-Mouse IgG (H + L) HRP Conjugate (1:8000, TransGen Biotech, HS201-01), respectively.