Figure 7.

DRAM2 co-localizes with clathrin vesicle adaptors AP-1 and AP-3 and affects their expression

(A) A significant reduction in protein expression was established for GARP component VPS53 and AP-1γ adaptor for CORD21-P2 and -P1 RO lysates, respectively. Scale bars represent 20 μm. Data are presented as mean +SEM (n = 3–4 different differentiation experiments each consisting of 48 ROs/sample) and normalized to the WT sample.

(B) RPE cell analysis by WB demonstrated a significant downregulation of clathrin transporter AP-3β in the lysates of CORD21-P2 RPE cells relative to isogenic controls. Total protein stain was used to visualize equal protein loading, and data were normalized to the WT sample. Scale bars represent 20 μm.Data are shown as mean +SEM (n = 3–4 different differentiation experiments each consisting of 2 wells of a 12-well plate of RPE cells/sample). Statistical comparisons for CORD21-P1vs -P1c and CORD21-P2vs-P2c are denoted by ^∗p <0 .05, ^∗∗p <0 .01.

(C) Clathrin (green) is detected in a dotty-like pattern across the entirety of the photoreceptor IS and can be seen to only partially co-stain with DRAM2 (red, left image). DRAM2 expression (red) strongly overlaps with that of transport vesicle proteins AP-1 (green, middle) and even more so with AP-3 (green, right) at the IS of ROs. Hoechst (blue) counterstains nuclei; white arrowheads show DRAM2 (red) co-localization with clathrin, AP-1, and AP-3 markers (green). These are representative examples from 15 ROs imaged from three different differentiation experiments. Scale bars represent 20 μm. Top panels scale bar, 20 μm; bottom panel scale bar, 10 μm.