Fig 2: Confocal laser microscopy: localization of hTRPV3 in X. oocytes overexpressing hTRPV3. a Anti-TRPV3 distinctly stained the cell membrane of hTRPV3 X. oocytes (n = 24), confirming successful expression and trafficking. b Controls (n = 20) injected with RNA-free water showed no signal after staining with the anti-TRPV3 antibody [39]

Fig 3: Expression of the Olmsted mutant G573S in HEK-293 cells. a HEK-293 cells expressing the hTRPV3 mutant G573S were immunostained 22 h after transfection (for details, see supplement part G (n = 26)). As in Fig. 3, DAPI (blue) was used to stain the cell nuclei. All three cells showed staining with the anti-TRPV3 antibody (red), with one cell showing clear trafficking of the protein to the cell membrane. Conversely, the HEK-293 cell that can be seen on the left showed strong cytosolic staining. Using transmitted-light microscopy, signs of a general structural degradation with partial loss of the cell membrane were observed in this cell. The GFP (green), which should be co-expressed as in Fig. 3, was too low for detection in all three cells. b Boxplots show the cell viability of hTRPV3, G573S, and control HEK-293 cells. Half of the passages were incubated with the TRP antagonist RR (n/N = 12/3). A significantly lower number of cells transfected with G573S (red) was found to be intact in comparison to cells transfected with hTRPV3 (blue) and controls (orange). Supplementing RR, cell viability was lower in the hTRPV3 and control groups and significantly higher in G573S. Different letters indicate significant differences determined using ANOVA on Ranks with Student–Newman–Keuls method. For the effect of RR, differences were determined using Mann–Whitney Rank Sum Test (*:p < 0.05; n.s.: not significant)

Fig 4: Immunoblots: detection of hTRPV3 in overexpressing HEK-293 cells, X. oocytes, and in human keratinocytes. Following the marker lane (M) and from left to right, lanes were loaded with protein samples extracted from G573S HEK-293 cells (1 µg; typical example of n = 8 replicates), control HEK-293 cells (10 ng; n = 19), hTRPV3 HEK-293 cells (10 ng; n = 14), hTRPV3 X. oocytes (5 µg; n = 7), control X. oocytes (5 µg; n = 8), G573S HEK-cells (1 µg; n = 11), control HEK-cells (20 ng; n = 15), hTRPV3 HEK-cells (20 ng; n = 10), hTRPV3 X. oocytes (1 µg; n = 7), control X. oocytes (1 µg; n = 7), and keratinocytes obtained from human skin equivalents (1 µg; n/N = 12/2). Staining with Anti-Strep, a band at the height of full-length TRPV3 (~ 95 kDa) can be seen in all overexpressing cells. At ~ 60 kDa, an additional weaker band was found in overexpressing HEK 293 cells only. Staining with Anti-TRPV3, all overexpressing cells showed the full-length band at ~ 95 kDa. X. oocytes expressed an additional band at ~ 80 kDa. Various breakdown products were stained at different heights in cells expressing the mutant G573S (see “Discussion” section). Control cells showed no staining. The keratinocyte protein extracted from human skin equivalents showed a weak band at ~ 95 kDa needing longer exposure times than normal in addition to various bands = 60 kDa

Fig 5: Immunohistological staining of bTRPV3 in Xenopus oocytes. a pGEM construct containing a Strep-tagged bTRPV3 sequence for in vitro transcription to cRNA. b and c Immunohistological staining of two oocytes 4 days after injection of b water or cbTRPV3 cRNA. Cells were stained with TRPV3 antibody and investigated using the same microscope settings. Only overexpressing oocytes c show staining of the cellular membrane with the TRPV3 antibody