Pitstop 2 Is a Potent Inhibitor of Clathrin-Independent Endocytosis (original) (raw)
Figure 3
Pitstop 2 inhibits SNAP-Tac internalization in transfected HeLa cells.
(A) HeLa cells transiently transfected with SNAP-Tac were preincubated for 10 min with either DMSO (control, top panels) or 20 µM pitstop 2 (bottom panels). Cells were then exposed to BG-S-S-594 and allowed to internalize for 30 minutes at 37°C in the presence or absence of pitstop 2. Cells were then washed with PBS, overlaid with Phenol Red-free DMEM containing 25 mM HEPES, and imaged live. Images in the left column were taken before the addition of TCEP, and represent total labeled SNAP-Tac protein (surface + internalized protein). Images of the same cells taken one min after TCEP treatment are shown in the right column, and represent the internalized pool of SNAP-Tac whose BG-S-S-594 label is protected from TCEP cleavage. (B) Percent internalized SNAP-Tac was calculated for individual cells displayed in (A) under either DMSO treatment or pitstop 2 treatment conditions. Small squares represent calculations of individual cells (12 cells measured for DMSO, 16 cells measured for pitstop 2 treatment). Horizontal bars represent the mean percent internalization for each group of cells (13.8% +/−1.6% for DMSO, 3.1% +/−1.1% for pitstop 2). This graph shows the quantification of a single endocytosis experiment. Data pooled from at least five matched sets (DMSO and pitstop 2) of comparable experiments and using two different pitstop 2 stocks demonstrated a similar inhibition of SNAP-Tac internalization in the presence of pitstop 2.