Figure 8. Detection of mRNA/protein for the key molecules for store operated calcium entry (SOCE) in H295R.

I) mRNA, Stromal interaction molecule 1 (STIM1), a Ca²⁺ sensor expressed on endoplasmic reticulum, calcium release-activated calcium channel protein 1 (Orai-1), a component for connection between STIM1 and Ca channels, and TRPC channels, a possible Ca channel for SOCE were examined. mRNA for STIM1 (A) Orai-1 (B), and subtypes of TRPC1 (C), C3 (D), C5 (E), and C6 (F) were identified in H295R. The lanes in each gel image show: molecular markers (M), three PCR products for the mRNA target primers (products in high, middle, and low annealing temperatures are indicated), and one using a GAPDH primer (G). Each value in parentheses indicates the molecular weight (bp) for the expected PCR product. The numbers on the three lanes of the targets represent the annealing temperatures used in the individual procedures. II) protein, Anti-human antibodies for STIM1, Orai-1, and TRIPC channels were used to confirm the expression of proteins for which mRNA was detected by PCR in figure 8 –I. The followings are the target proteins and their predicted molecular weights; a–1: STIM1 (84 kDa), a–2: STIM1 with control peptide (0.2 µg/mL) in the primary antibody reaction, b: Orai-1 (52 kDa), c: TRPC1 (83 kDa), d: TRPC3 (97 kDa), e: TRPC5 (110 kDa), f: TRPC6 (100 kDa). Molecular sizes (kDa), estimated by pre-stained weight marker, are shown on the left side of each membrane image. Each arrow head indicates the signal band that is clear and nearest to the predicted size of the target protein.