For Fluo-4 AM Ca2+ imaging, HEK293 cells transfected with GST-TRPV1 or primary cultured DRG neurons were loaded with 5 μM Fluo-4 AM (Invitrogen, USA) for 40 min at 37°C and then washed three times and left in Hanks’ balanced salt solution (HBSS) without phenol red (GE Healthcare, USA). Cells were then transferred at 37°C to allow membrane protein internalization or 4°C for control for 15 min. After being balanced at room temperature for more than 30 min, cell images were acquired every 5 s with an inverted Leica TCS SP8 microscope (Germany) with an APO 25× objective (water, NA 0.95) and a 488-nm argon laser. The real-time fluorescence intensity was analyzed by Leica LAS X software.

For Fura-2 AM Ca2+ imaging, after application of TAT-S45 peptide or TAT-S45A peptide (10 μg) for 4 hours, HEK293 cells were subjected to Fura-2 AM-based Ca2+ imaging experiments as described previously (19). Cells were loaded with 5 μM Fura-2 AM (Biotium) for 30 min at 37°C and then washed three times and left in HBSS without phenol red (GE Healthcare). For Ca2+ imaging, an inverted fluorescent microscope equipped with a 340- and 380-nm excitation filter changer (Olympus) with MetaFluor software was used. The F340/F380 ratio was acquired every 5 s. TRPV1 activation was evoked by the addition of 5 μM capsaicin. Cells that failed to respond to 25 mM KCl were discarded from the analysis. Cells derived from at least three separate isolations were analyzed.

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