Figure 5.

PDI interactions with ALLC are altered by 147 treatment. (A) Representative immunoblot of PDIA1 and PDIA4 in ALLC immunopurifications from ALMC-2 cells treated for 5 hours with vehicle (Veh) or 147 (10 µM). Inputs from this experiment are shown in supplemental Figure 5E. (B) Quantification of PDIA1 and PDIA4 from immunoblots as shown in panel A. The recovery of each PDI was normalized to the recovery of ALLC under each condition, allowing accurate evaluation of the interaction between these 2 proteins. Error bars show standard error of the mean (SEM) for >5 independent experiments. (C) Representative fluorescence gel showing the covalent modification of proteins in ALMC-2 cells treated for 18 hours with 147-alkyne (10 µM), RB-11-ca (30 µM), or KSC-34 (30 µM). Click chemistry was used to incorporate a TAMRA fluorophore onto the alkyne contained in each of these molecules. Cells treated with 147 are shown as control. PDI bands were assigned based on previous mass spectrometric analysis of SDS-PAGE bands excised from identical gels of 147-alkyne–treated ALMC-2 cell lysates.³⁹ (D) Normalized quantification of PDIA1, PDIA4, and PDIA6 labeling from gels as shown in panel C. Error bars show SEM for 4 replicates across 2 independent experiments. *P < .05, **P < .01, ***P < .005 (paired Student t test).