Cell lytic b cell lysis reagent

Five hundred mL cultures of C. bescii strains (JWCB017, 049, and 054) were grown to mid-log phase at 75 °C, harvested by centrifugation at 6000×g at 4 °C for 15 min and resuspended in Cell-Lytic B cell lysis reagent (Sigma-Aldrich, St. Louis, MO, USA). Cells were lysed by a combination of 4× freeze-thawing and sonication on ice. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin as the standard. Cell free extracts (75 µg) were electrophoresed in 4–15 % gradient Mini-Protean TGX gels, which were either stained using Coomassie blue or were transferred to PVDF membranes (ImmobilonTM-P; EMD Millipore, Billerica, MA, USA) using a Bio-Rad Mini-Protean 3 electrophoretic apparatus. The membrane was then probed with His-tag (6xHis) monoclonal antibody (1:5000 dilution; Invitrogen, Grand Island, NY, USA) using the ECL Western Blotting substrate Kit (Thermo Scientific, Waltham, MA, USA) as specified by the manufacturer.

Five hundred mL cultures of C. bescii were grown to mid-log phase at 65, 70 or 75°C, harvested by centrifugation at 6,000×g at 4°C for 15 min and resuspended in Cell-Lytic B cell lysis reagent (Sigma-Aldrich, St. Louis, MO, USA). Cells were lysed by a combination of 4X freeze-thawing and sonication on ice. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin as the standard. Cell free extracts (80 µg) were electrophoresed in 4–15% gradient Mini-Protean TGX gels, which were either stained using Coomassie blue or transferred to PVDF membranes (ImmobilonTM-P; EMD Millipore, Billerica, MA, USA) using a Bio-Rad Mini-Protean 3 electrophoretic apparatus. The membrane was then probed with His-tag (6xHis) monoclonal antibody (1:5,000 dilution; Invitrogen, Grand Island, NY, USA) using the ECL Western Blotting substrate Kit (Thermo Scientific, Waltham, MA, USA) as specified by the manufacturer.

Strains expressing the ApdA reporter were grown in 100 mL of LB with 20 μM bortezomib until OD₆₀₀ = 0.5 and harvested by centrifugation. The pellet was frozen at −80 °C and thawed in 2× CellLytic B-cell lysis reagent (Sigma) and 0.2 mg mL⁻¹ lysozyme for 10 min. The lysate was clarified by centrifugation for 30 min at 20,000×g at 4 °C. In total, 50 μL of Strep-tactin Sepharose beads (IBA) were added to the supernatant and samples were incubated at 4 C for 1 h. The beads were washed four times with IP wash buffer (20 mM Tris pH 8.0, 100 mM NH₄Cl, 0.4% Triton X-100, 0.1% NP-40) for 5 min at 4 °C. Protein was eluted from the beads by shaking at 4 °C in elution buffer (20 mM Tris pH 8.0, 100 mM NH₄Cl, 5 mM desthiobiotin) for 1 h. Then, 36 μl of the immunoprecipitated sample was reduced with 100 mM DTT in 100 mM triethylammonium bicarbonate (TEAB) buffer at 58 °C for 55 min and then the pH was adjusted to 8.0. The samples were alkylated with 200 mM iodoacetamide in 100 mM TEAB buffer in the dark at room temperature for 15 min. Proteins were pelleted and resuspended in 50 mM TEAB and proteolyzed with 15 ng μL⁻¹ of LysC (Wyco) at 37 °C overnight. Peptides were desalted on Oasis u-HLB plates (Waters), eluted with 60% acetonitrile (ACN)/0.1% trifluoracetic acid (TFA), dried and reconstituted with 2% ACN/0.1% formic acid.