Ecl super signal west femto substrate reagent

1 × 10⁶ MCF-7 Y537S cells were cultured on a 35-mm dish. Cells were treated with 8 Gy of IR after pre-treatment with vehicle or OTX015 (1 μM) for 1 hour. 24 hours after IR treatment, cells were washed with ice-cold PBS and lysed with modified RIPA lysis buffer containing 50 mM Tris-HCl pH 7.5, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, complete protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche), resolved by SDS-PAGE, and transferred onto a PVDF membrane. The membrane was immunoblotted with antibodies against LC3-I/II (1:1000 dilution; Cat #12741, Cell Signaling Technology). LC3-I/II proteins were detected using ECL super signal west femto substrate reagent (Thermo Scientific). The experiment was repeated thrice and a representative image was shown.

THP1 cells, BMDM, HEK293T, A549, and Calu3 cells were lysed in ice-cold RIPA lysis buffer containing complete protease inhibitor and phosphatase inhibitor cocktails (Roche), resolved by SDS-PAGE, and transferred onto a PVDF membrane. The membranes were immunoblotted with antibodies against Phospho-NF-κB p65 (3033, Cell Signaling), Phospho-IκBα (9246, Cell Signaling), IκBα (4812, Cell Signaling), Phospho-ERK (4370, Cell Signaling), ERK (4695, Cell Signaling), Phospho-JNK (4668, Cell Signaling), Phospho-AKT (4060, Cell Signaling), Phospho-STAT3 (9145, Cell Signaling), SARS-CoV-2 S (GTX632604, GeneTex), and β-actin (A2228, Sigma-Aldrich). Immunoreactive protein bands were detected using ECL super signal west femto substrate reagent (Thermo Fisher Scientific).

Mouse liver tissues or cultured cells were homogenized in RIPA lysis buffer containing complete protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche), resolved by SDS-PAGE, and transferred onto a PVDF membrane. The membranes were immunoblotted with antibodies against ERK (4695, Cell Signaling), Phospho-ERK (4370, Cell Signaling), JNK (9252, Cell Signaling), Phospho-JNK (4668, Cell Signaling), Phospho-cJun (3270, Cell Signaling), p38 (8690, Cell Signaling), Phospho-p38 (9215, Cell Signaling), AKT (9272, Cell Signaling), Phospho-AKT (4060, Cell Signaling), β-catenin (8480, Cell Signaling), Phospho-β-catenin (9565, Cell Signaling), Phospho-STAT3 (9145, Cell Signaling), Phospho-NF-κB p65 (3033, Cell Signaling), cMyc ( 5605, Cell signaling), Cyclin d1 (2978, Cell signaling), Anti-FlagM2 (F1804, Sigma-Aldrich), NLRP12 (Aviva Systems Biology, #OAAB04256), and β-actin (A2228, Sigma). Finally, immunoreactive proteins were detected using ECL super signal west femto substrate reagent (Thermo Scientific).

Hormone-deprived cells were lysed with modified RIPA lysis buffer containing 50 mM Tris-HCl pH 7.5, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, complete protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche), resolved by SDS-PAGE, and transferred onto a PVDF membrane. The membranes were immunoblotted with antibodies against BRD4 (1:1000 dilution; Cat #13440, Cell Signaling Technology) and β-actin (1:4000 dilution; Cat #3700, Cell Signaling Technology). Immunoreactive proteins were detected using ECL super signal west femto substrate reagent (Thermo Scientific). All blots were derived from the same experiment and were processed in parallel.

MCF-7 cells were transfected with FLAG-BRD4 and HA-ERα (WT, Y537S, or D538G). Forty-eight hours after transfection, cells were homogenized in modified RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.5), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, complete protease inhibitor cocktail, and phosphatase inhibitor cocktail (Roche). Immunoprecipitation was performed with mouse anti-FLAG antibody (MilliporeSigma, F3165), resolved by SDS-PAGE, and transferred onto a PVDF membrane. The membranes were immunoblotted with antibodies against HA (Cell Signaling Technology, catalog 3724) and anti-FLAG (MilliporeSigma, catalog F3165). Immunoreactive proteins were detected using ECL SuperSignal West Femto substrate reagent (Thermo Fisher Scientific). For endogenous immunoprecipitation and immunoblotting experiments, similar protocol was employed in native MCF-7 Y537S cells, and immunoprecipitation and immunoblotting were performed with antibodies against endogenous ERα (Cell Signaling Technology, catalog 8644) and BRD4 (Cell Signaling Technology, catalog 13440).