Calcein red am

HeLa cells, either untransfected or transfected with GFP-Cx37-WT, alone or together with GFP-Cx37-G316S or GFP-Cx37-P258S expression vectors for 24 h, were loaded for 30 min at 37°C with 5 µM calcein-red AM (Invitrogen; Thermo Fisher Scientific, Inc.) in PBS. Dye transfer was then monitored with a Zeiss LSM 510 confocal microscope, as previously described (18 (link)). Randomly selected cells were bleached with a 543-nm laser pulse and the recovery of fluorescence intensity was monitored by analyzing data obtained prior to bleaching, just after bleaching and 30 min after photobleaching using Zeiss LSM software (version 2.01). The percentage of fluorescence recovery was determined by normalizing all values to the intensities prior to photo-bleaching set as 100% and subtracting the normalized value for cells just after photobleaching from the normalized value obtained at 30 min after photobleaching. At least 20 cells were analyzed for each set of experimental conditions and at least three separate experiments were performed.

Calcein-red AM was purchased from Invitrogen (Thermo Fisher Scientific, Inc., Illkirch, France), while rabbit anti-giantin (1:1,000 dilution; ab80864), rabbit anti-early endosome antigen 1 (EEA1; 1:1,000 dilution; ab2900) and mouse anti-lysosome-associated membrane protein 2 (LAMP2; 1:100 dilution; ab25631) (all from Abcam, Cambridge, UK) antibodies were purchased from Covance (Princeton, NJ, USA), Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and BD Pharmingen (San Jose, CA, USA), respectively, and goat anti-rabbit and anti-mouse secondary antibodies conjugated to Alexa Fluor 647 (cat. nos. 115-605-146 and 111-605-144) were obtained from Jackson ImmunoResearch (West Grove, PA, USA). GFP-Rab5 probe was a gift from M. Zerial Laboratory (Dresden, Germany).