Figure 2. FIGURE 2: The IGP family are developmentally regulated ER proteins.

(A) Copy numbers of IGP48 39, IGP40 40 and IGP34 34 mRNAs measured by qRT-PCR, in different life cycle stages, normalised to long slender BSF mRNA levels at 1.0. Error bars denote standard errors of the mean from triplicate measurements on independent RNA samples. Western blot of trypanosome whole cell lysates using anti-IGP48 affinity-purified antisera raised against E. coli-expressed recombinant protein at 1:100 dilution. The blot was re-probed for PAD1 (protein associated with differentiation 1), and which is specifically unregulated in the stumpy bloodstream form, to validate the short stumpy lysate. Rightmost; whole cell lysate probed with anti-IGP48 antisera to validate specificity.

Abbreviations: BSF (LS), long slender bloodstream form; BSF (SS), short stumpy bloodstream form and PCF, procyclic culture form.

(B) Intracellular localisation of IGP48-HA and IGP40-HA in BSF cells under permeabilised conditions, and detected with anti-HA antibody (red). Top panel: co-staining with anti-TbBiP (green). Lower panels: co-straining with anti-TbRabX2 and anti-p67 (green) using confocal microscopy. Bar = 2µm. Inset: expression of IGP48-HA and IGP40-HA in 427 BSF cells detected by Western blotting using anti-HA antibody.

(C) Digestion of IGP48 with PNGase F or Endo H, or treatment with tunicamycin results in a large molecular weight shift. IGP48 was detected in fractionated lysates using anti-HA antibody.

(D) Turnover of IGP48 and IGP40. Quantification of anti-HA reactivity in lysates of cells expressing IGP48-HA and IGP40-HA following inhibition of protein synthesis with cycloheximide. Error bars represent the standard deviation and values were normalised against a loading control, BiP (n = 3).