Fig. 1
From: PTHrP intracrine actions divergently influence breast cancer growth through p27 and LIFR
Validation of plasmids expressing specific PTHrP domains. (A) Pthlh overexpression construct design and validation in MCF7 cells by (B) western blot for the C-terminal HA-Tag and qPCR for the (C) mid-region, (D) nuclear localization sequence (NLS), and (E) C-terminal domain. MSCV = control, FLSEC = full-length secreted, DNLS = NLS deleted, DNLS + CTERM = NLS and C-terminal domain deleted. Predicted molecular weights: FLSEC PTHrP (-36-139aa) = 21.2kD, DNLS PTHrP (-36-67aa)…(95-139aa) = 18kD, DNLS + CTERM PTHrP (-36-67aa) = 12.8kD. GAPDH = loading control. (F) Immunocytochemical staining for HA-Tag (green) and DAPI (blue). All panels = 100X and scale bars = 25 μm. (G) Secreted PTHrP (1-34aa) levels measured by ELISA from conditioned media of cells described in (A). (B-E & G) n = 3 independent biological replicates. Graphs represent mean ± SEM. (C) **p < 0.001 vs. MSCV or *p < 0.05 vs. FLSEC by one-way ANOVA with multiple comparisons. (D) **p < 0.001 vs. FLSEC by one-way ANOVA with multiple comparisons. (E) **p < 0.001 vs. DNLS by one-way ANOVA with multiple comparisons