Lysates of the patients P1 and P2 and control fibroblasts as well as HeLaM cells were used for immunoblot analysis with anti-human VPS11 (WH0055823M1; Sigma-Aldrich), anti-human VPS41 (sc-377 118; Santa Cruz) or antibodies to VPS33A, VPS 18, HA and actin as described previously (10 ). In addition, lysates from the transfected HeLaM cells were subjected to immunoblot analysis with anti-HA (
HA.11, MMS-101R; Covance) and
anti-actin (A2066; Sigma-Aldrich) antibodies. In experiments to examine the effects of the proteasome inhibitor MG-132 on HeLaM cells stably expressing VPS33A
WT-HA or VPS33A
R498W-HA, immunoblotted VPS33A bands were quantified by densitometry using ImageJ software, normalized to actin bands and fold changes in VPS33A concentration calculated relative to no incubation with MG-132. In each separate experiment, the effect of MG-132 on both VPS33A
WT-HA and VPS33A
R498W-HA was measured. A paired student
t-test was used to calculate
P-values.
Pavlova E.V., Shatunov A., Wartosch L., Moskvina A.I., Nikolaeva L.E., Bright N.A., Tylee K.L., Church H.J., Ballabio A., Luzio J.P, & Cox T.M. (2019). The lysosomal disease caused by mutant VPS33A. Human Molecular Genetics, 28(15), 2514-2530.