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. 2007 Sep 6;26(19):4177–4188. doi: 10.1038/sj.emboj.7601844

Figure 4.

Figure 4

ERGIC-53 mediates ERp44 localization in the early secretory pathway. (A) HeLa cells transiently transfected with vectors driving the expression of wt ERGIC-53 (a–c) or the ER-localized mutant ERGIC-53 KKAA (d–f) were co-stained with antibodies specific to ERp44 and ERGIC-53. Images were taken with a fluorescence microscope and analyzed with deconvolution techniques. In each panel, boxes show a higher magnification of the indicated areas. Arrows indicate examples of colocalizing structures. Mutant ERGIC-53 KKAA acts as a localization dominant negative and causes the ER accumulation of endogenous ERGIC-53 molecules as well (Vollenweider et al, 1998 and data not shown); note that endogenous ERp44 is in part delocalized with the mutant lectin, as demonstrated by the diffuse pattern shown in panel d and by the colocalization of the two molecules (f). However, some ERp44 is able to reach the ERGIC region (not shown). Bar=10 μm. (B, C) HeLa cells were transiently transfected with HA-ERp44ΔRDEL alone (ctrl, empty square) or with wt ERGIC-53 (53 wt, filled circles) or the ER-localized KKAA mutant (KKAA, filled triangles). Forty-eight hours after transfection, cells were pulse-labeled and chased for the indicated times as described in legend to Figure 2. Cell lys and SN were IP with anti-HA (to isolate ERp44), resolved by reducing SDS–PAGE and subjected to autoradiography (B). Panel C shows densitometric analyses of lys, performed as in legend to Figure 2 (average of four independent experiments, ±s.e.m.); signals were normalized relative to a stable background (see * in panel B). The overexpression of either wt or KKAA ERGIC-53 inhibits ERp44ΔRDEL secretion. (D) HeLa cells were subjected to RNAi for ERGIC-53 (53i, filled diamonds) or treated with control duplexes (luc2i, empty triangles), and then transiently transfected with HA-ERp44ΔRDEL, as indicated. Seventy-two hours from RNAi (48 h from transfection), cells were pulse-labeled, chased and analyzed as described for Figure 2. In the absence of ERGIC-53, ERp44ΔRDEL is secreted more rapidly (average of three independent experiments, ±s.e.m.). As a control of the efficiency of RNAi, see Figure 5B.