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. 2019 Jul 17;25(13-14):1013–1022. doi: 10.1089/ten.tea.2018.0182

FIG. 3.

FIG. 3.

NHE3 expression varies with substrate stiffness. (A) Expression levels of SLC9A3 (NHE3) were determined by qPCR in primary HREC cultured on soft (0.5 kPa) and stiff (50 kPa) hydrogels. Cells were harvested at short (10 days) and long (4 weeks) time points. (B) NHE3 protein levels were determined by Western blot analysis. Protein was isolated from HREC on soft and stiff hydrogels, cultured in parallel with cells used for qPCR analysis. Twenty microgram of total protein was loaded per lane, and blots were probed with anti-NHE3 antibody (sc-16103 @ 1:1000). GAPDH was used as a loading control. (C) NHE3 (sc-16103 primary @ 1:200 with Alexa Fluor 555 secondary, red) and F-actin (Alexa Fluor 488 Phalloidin, green) staining of HRECs cultured on soft (0.5 kPa) and stiff (50 kPa) hydrogels for 4 weeks. Inset images show samples of the same images at double magnification. HREC, human renal epithelial cell.