Calcineurin

Sorted BM B220⁺CD43⁺ pro-B and B220⁺CD43⁻ pre-B cells from WT or Rag1^−/− mice or B220⁺IL-7R⁺ pro-B and B220⁺IL-7R⁻pre-B cells from WT mice were immunostained with NFATc1, NFATc2 (both from ImmunoGlobe), or NFATc3 (Santa Cruz) Abs following a previously published protocol.^{39 (link)} Counterstaining with 4,6-diamidino-2-phenylindole was performed to confirm nuclear staining. Image acquisition and data analysis were performed using a TCS SP2 Leica confocal microscope and software.
Whole-cell protein extracts from the WT pro- and pre-B cells were prepared as mentioned previously.^{40 (link)} Fifty micrograms of total protein was analyzed in an 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to immunoblotting for detection of calcineurin (Cell Signaling Technology) and PLCγ2 (Santa Cruz) levels. Actin was used as a loading control.

For Western blotting experiments, cells were lysed in RIPA buffer containing 50 mM of Tris/HCl, (pH of 7.4) with1% Nonidet P-40, 0.5% Nadeoxycholate, 150 mM of NaCl, 1 mM of EDTA, 1 mM of PMSF, 1 mM of Na3VO4, 1 mM of NaF, and protease inhibitor (Sigma). Then cells were separated via SDS/PAGE, followed by being transferred to a 0.45-mm-pore PVDF membrane (MilliporeSigma, Burlington, MA, USA). Later, the membrane was blocked with 5% milk for 1 h, incubated with the relevant primary antibodies at 4°C overnight, and was then incubated with horseradish-peroxidase-conjugated secondary antibodies (1:2000) at room temperature for 1 h. An ECL Kit (MilliporeSigma) was used to detect the immunoreactive bands. The primary antibodies: Carabin (1:500) (Cat#PA5-20394, Thermo Scientific), Calcineurin (1:1000) (Cat#2614S, Cell Signaling, Danvers, MA, USA), NFAT (1:1000) (Cat#5861S, Cell Signaling), Ras (1:1000) (Cat#3339S, Cell Signaling), ERK (1:1000) (Cat#4696S, Cell Signaling) and p-ERK (1:500) (Cat#8544S, Cell Signaling). Antibodies used in this study for WB were listed in Supplementary Materials.

Protein extraction was performed as previously described 52 (link). Cytosolic and nuclear proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific), following the manufacturer's protocol. Equivalent level of protein was resolved by 10% SDS-PAGE gel, transferred onto polyvinylidene fluoride (PVDF) membranes and then blocked with 5% bovine serum albumin (BSA). Membranes were incubated, overnight at 4 °C, with primary antibodies against MVP (1:1000, Santa Cruz), NFATc1 (1:500, Santa Cruz), PU.1 (1:1000, Cell Signaling Technology), c-Fos (1:1000, Abcam), MITF (1:1000, Abcam), CTSK (1:1000, Santa Cruz), p-NFATc1 (1:1000, Invitrogen), Calcineurin (1:1000, Cell Signaling Technology), H3 (1:1000, Cell Signaling Technology), Lamin B (1:1000, Cell Signaling Technology) and GAPDH (1:8000, Bioworld), and then washed with TBST, incubated with secondary antibodies, and visualized by enhanced chemiluminescence. Semi-quantitative measurements were performed using Image J software (National Institutes of Health, USA).

The procedure was adapted from our previous studies.13 Total proteins from the tissues and cells were extracted with lysis buffer including 1% protease inhibitor solution. Briefly, the protein concentrations were determined by BCA protein assay kit. Protein samples (100 mg) were separated by 10% SDS‐PAGE and transferred to nitrocellulose membranes and then blocked with 5% nonfat milk. The membranes were probed overnight at 4°C with the following antibodies: TRPV3 (1:200; Alomone labs, Jerusalem, Israel), p‐CAMKII and CAMKII (1:1000; Cell Signaling Technology, Boston, MA, USA), calcineurin (1:1000; Cell Signaling Technology), β‐MHC (1:3000; Sigma), NFATc3 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), BNP (1:100; Santa Cruz Biotechnology), Hsp70 (1:300; Boster Inc., Wuhan, China), β‐actin (1:2000; Zhong Shan‐Golden Bridge Biological Technology, Beijing, China), GAPDH (1:2000; Zhong Shan‐Golden Bridge Biological Technology). β‐actin or GAPDH was as a loading control. After washing three times, members were incubated with a horseradish peroxidase‐conjugated secondary anti‐rabbit/mouse/goat IgG for 2 hours at room temperature. Proteins were visualized using ECL (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and collected by using the Bio Imaging Systems (UVP Inc., Upland, CA, USA).