Fig. 2. The Bat3 complex mediates substrate capture by TRC40.

a, Translation extracts were passed over anti-GFP (control), anti-Bat3, or anti-TRC40 affinity resins and different amounts of each depleted lysate analyzed by immunoblotting. b, Substrate capture assay using either total cytosol, or cytosol immunodepleted (‘Δ’) with the indicated affinity resins. In this assay (see Sup. Fig. S1), radiolabeled Sec61β RNCs are released with puromycin and capture by TRC40 is assessed by crosslinking. The portion of gel showing the TRC40-Sec61β crosslink is shown. Failure of TRC40 to capture substrate typically results in capture by a 38 kD protein (p38). The ‘addback’ lanes are Bat3-depleted cytosol replenished with affinity purified Bat3 complex (prepared using an anti-TRC35 resin; Sup. Fig. S8) at three concentrations spanning that present in the original cytosol. The ‘mock’ addback sample was prepared in parallel, but employed an irrelevant affinity resin (anti-GFP) in lieu of TRC35 affinity resin (Sup. Fig. S8). A reaction lacking crosslinker (XL) is shown in the first lane. Aliquots of each reaction (prior to crosslinking) were also analyzed by immunoblot against Bat3 and TRC40 to document their relative amounts in the reactions.