Pierce radioimmunoprecipitation assay buffer

siRNA-transfected cells were extracted and centrifuged. Cell pellets were lysed in Pierce radioimmunoprecipitation assay buffer (89900, Thermo Fisher Scientific) with Protease Inhibitor Cocktail Tablets (11836170001, Roche) and PhosSTOP Phosphatase Inhibitor Cocktail Tablets (04906845001, Roche). The protein concentration in each sample was measured using the Pierce BCA Protein Assay Kit (23227, Thermo Fisher Scientific). Laemmli Sample Buffer (1610747, Bio-Rad) was used to dilute samples to 3.5 μg/μl. Thirty-five micrograms of total protein was added in each lane of 4 to 15%, 15-well gradient gels (4561086, Bio-Rad). The gels were run for 35 min and the proteins were transferred to nitrocellulose (Bio- Rad) at 100 V for 45 min. The membrane was blocked in 5% milk for 1 hour, incubated overnight in primary antibodies against Vimentin (ab92547, Abcam; 1:1000), Nesprin3 (ab74261, Abcam; 1:500), Lamin A/C (2032S, Cell Signaling Technology; 1:1000), NHE-1 (SC-136239, Santa Cruz Biotechnology; 1:1000), TRPV4(ab39260, Abcam; 1:1000), P38 (SC-535, Santa Cruz Biotechnology; 1:1000), and glyceraldehyde-3-phosphate dehydrogenase (ab181602, Abcam; 1:1000). Blots were incubated with secondary antibodies against the primary for 1 hour and imaged using a LI-COR Odyssey imaging system (LI-COR Biotechnology).

Blocks containing forebrain cortex, dorsal hippocampus and amygdala were dissected from brains using a mouse brain matrix (Zivic Instruments). Then, 1 mm slices were cut in Paxinos and Franklin's Mouse Brain Atlas²⁴ coordinates bregma 1.10–0.10 and bregma −1.22 to 2.18 approximately. Blocks were homogenized in Pierce radioimmunoprecipitation assay buffer (Thermo Scientific), and protein concentrations were measured using Pierce bicinchoninic acid protein assay kit (Thermo Scientific), and adjusted to 0.8 μg/μl. An automated western assay was then performed, using 12–230 KDa separation module (Protein Simple), in a WES system (Protein Simple), following protocol from manufacturer, using antibodies for GR (no. 3660, 1:20; Cell Signaling Technology; RRID:AB_11179215), MR (monoclonal antibody kindly provided by Professor Gomez‐Sanchez,²⁵ clone1D5, 1:10), and glyceraldehyde 3‐phosphate dehydrogenase (no. 5174, 1:20; Cell Signaling Technologies; RRID:AB_10622025). The image was analysed in Compass for Simple Western software (Protein Simple).

The 5001 cell line stably expressing hCaSR was starved in serum-free DMEM medium supplemented with 0.2% (w/v) BSA overnight, followed by washing with Hanks’ balanced salt solution (HBSS) three times and a subsequent 10-min HBSS incubation in the morning of the second day. To induce ERK1/2 phosphorylation, varying concentrations of [Mg²⁺]_o (0 to 50 mM) or [Ca²⁺]_o (0 to 30 mM) with or without 0.5 mM TNCA were added to cells and incubated for 10 min at 37°C. The cells were then lysed with Pierce radioimmunoprecipitation assay buffer (Thermo Scientific). Total protein concentration was measured using the Bio-Rad assay. Lysates containing 100 μg of total protein were loaded onto 4 to 20% gradient SDS-PAGE gels for separation. After electrophoresis, proteins on the gel were transferred to nitrocellulose membranes and were further analyzed by Western blotting. Anti–phospho-p44/42 ERK (1:1000 dilution) and anti-p44/42 (1:2000) polyclonal antibodies were utilized as probes to detect the phospho-ERK1/2 and total ERK1/2, respectively. A chemiluminescent detection method (AP Conjugate Substrate Kit, Bio-Rad) was applied to detect phospho-ERK1/2 and total ERK1/2. The respective bands on Western blots were evaluated by densitometry. The EC₅₀ of [Mg²⁺]_o- or [Ca²⁺]_o-dependent responses were obtained by fitting the [Mg²⁺]_o or [Ca²⁺]_o concentration-response curves with the Hill equation (Eq. 1).