Skip to main content
. 2012 Aug 19;21(23):5019–5038. doi: 10.1093/hmg/dds343

Figure 2.

Figure 2.

OCRL-depleted HeLa cells exhibit an increased plasma membrane level and internalization of MPR300. (A) HeLa cells were treated with OCRL-specific siRNAs for 72 h. GFP-specific siRNA was used as a control. Depletion was monitored by immunoblotting with an anti-OCRL antibody, and an anti-tubulin antibody was used as loading control. The blot shows a representative result obtained upon depletion with one of three different OCRL-specific siRNAs applied in this work. Depletion efficiency of all siRNAs was comparable with >90% of OCRL depleted after 72 h. (B) GFP and OCRL siRNA-treated cells were biotinylated at 4°C and subjected to streptavidin agarose as described in Materials and Methods. Aliquots of total cell lysates (2%; T) and precipitated cell surface proteins (20%; CS) were analysed by MPR300 immunoblotting. A representative immunoblot of four independent experiments is shown. (C) Levels of MPR300 were quantified by scanning densitometry, and cell surface MPR300 was normalized to total MPR300. Each bar is the mean ± SD of four independent experiments. **P = 0.003 (two-tailed Student's t-test). To exclude off-target effects, three different siRNAs against OCRL were independently applied. (D–F) GFP and OCRL siRNA-treated cells were biotinylated at 4°C and either harvested (CS) or incubated for 2 (I2) or 5 (I5) min at 37°C. Remaining biotin at the cell surface was removed using glutathione washes at 4°C, and the internalized biotinylated proteins were precipitated with streptavidin agarose followed by MPR300 immunoblotting. Levels of MPR300 were quantified by scanning densitometry and normalized to total MPR300. (D) A representative immunoblot is shown. Internalized MPR300 is expressed as percentage of cell surface MPR300 ± SD of six independent experiments. (E) Internalization rates were calculated as percentage of biotinylated cell surface receptors which are internalized per minute. Each bar is the mean ± SD of six independent experiments. (F) Internalized MPR300 in OCRL-depleted cells is expressed as percentage of control cells (white square GFP siRNA, black square OCRL siRNA). Each bar is the mean ± SD of at least six independent experiments. I2 (incubation for 2 min at 37°C): *P = 0.04; I5 (incubation for 5 min at 37°C): *P = 0.02 (two-tailed Student's t-test).