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. 2019 Oct 17;17(10):587. doi: 10.3390/md17100587

Figure 4.

Figure 4

Tv1 co-localizes with TRP Channels. (a) Intensities of TRPV6 and TRPC6 channel expressions are compared for 1MEA and BNL.CL2 cells at 800nm channels after normalizing at 700 nm channel for cell-tag. TRPV6 and TRPC6 in 1MEA have significantly increased levels (p < 0.05) of cells compared to BNL cells. (b) Co-localization analysis of Tv1 peptide labeled with fluorescein dye (Tv1-Fam) with TRPV6 and TRPC6 channels in 1MEA cells using MOC (manders overlap coefficient). Y-axis indicates incubation of specific channel, TRPV6 or TRPC6. The X-axis indicates the filters used for imaging, from left to right: DAPI, GFP, DAPI & CY5, DAPI, GFP, and Cy5. High MOC values (0.80 ± 0.01 and 0.9 ± 0.05 for TRPC6 and TRPV6, respectively) confirmed Tv1 co-localizes with these channels. (c) A specific blocker for TRPV6 channel, truncated SorC labeled with rhodamine (TruncSorC-Rho), was used to demonstrate colocalization as an indicator of activity. Both TruncSorC-Rho of Tv1-Fam with TRPV6 channels in its presence. High MOC for TruncSorC-Rho (0.88 ± 0.04) and Tv1-Fam (0.9 ± 0.05) with the channels indicate colocalization. Fluorescein labeled non-specific peptide ACP-Fam, probed with channel antibodies (TRPC6 and TRPV6) was used to examine non-specific green fluorescence and no colocalization was detected. Dyes and channels used: DAPI-staining nucleus for the presence of cells, GFP- green channel for the presence Tv1-Fam, Rho channel for the presence of Rhodamine labeled TruncSorC-Rho peptide, Cy-5 channel for the presence of ion channels as secondary antibodies used were cy-5 labeled.