Edta free protease inhibitor tablet

Cell lines: Human breast cancer cell line (MCF-7), TNBC (MDA-MB-231), and murine metastatic breast cancer cell line (4T1).
Reagents: Dulbecco’s modified eagle medium (DMEM) powder, fetal bovine serum (FBS), penicillin streptomycin, trypLE Express without phenol red and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) from Gibco BRL (Carlsbad, CA, USA). DMEM media, calcium chloride dihydrate (CaCl₂·2H₂O), sodium bicarbonate (NaHCO₃), potassium phosphate monobasic (KH₂PO₄), sodium phosphate dibasic heptahydrate (H₁₅Na₂O₁₁P), bovine serum albumin (BSA), skim milk powder, tris (hydroxmethyl)amino-methane, phosphate inhibitor cocktail 2, EDTA-free protease inhibitor tablets, dimethyl sulphoxide (DMSO), and thiazolyl blue tetrazolium bromide (MTT), methanol, and trypan blue (0.4%) from Sigma-Aldrich (St. Louis, MO, USA). Sodium chloride (NaCl) and potassium chloride (KCl) from Fisher Scientific (Loughborough, Leicestershire, UK). Sodium dodecyl sulfate (SDS) and Restore PLUS Western blot stripping buffer from Thermo Fisher Scientific (Rockford, IL, USA). Triton C-100, glycine, blotting-grade blocker, Clarity Western ECL substrate, tween-20, bromophenol blue, quickstart Bradford 1X dye reagent, quickstart bovine serum albumin (BSA) standard set, dithiothreitol (DTT), and mini-protean TGX gels from Bio Rad (Hercules, CA, USA). Validated siRNAs from Qiagen (Valencia, CA, USA).

B56γ cell pellets were resuspended in ice-cold lysis buffer (50 mM Tris pH 8.0, 0.5 M NaCl, 5 mM imidazole, 0.1% Triton X-100 containing EDTA-free protease inhibitor tablet [Sigma]), lysed by high-pressure cell homogenization (Avestin C3 Emulsiflex) and centrifuged (35,000 xg, 40 min, 4°C). The supernatant was loaded onto a HisTrap HP column (GE Healthcare) pre-equilibrated with Buffer A (50 mM Tris pH 8.0, 500 mM NaCl and 5 mM imidazole) and was eluted using a linear gradient of Buffer B (50 mM Tris pH 8.0, 500 mM NaCl, 500 mM imidazole). Fractions containing the protein were pooled and dialyzed overnight at 4°C (50 mM Tris pH 8.0, 500 mM NaCl) with TEV protease to cleave the His₆-tag. The cleaved protein was incubated with Ni²⁺-NTA beads (GEHealthcare) and the flow-through collected. The protein was concentrated and purified using size exclusion chromatography (SEC; Superdex 75 26/60 [GE Healthcare]) pre-equilibrated in ITC buffer (50 mM sodium phosphate pH 7.5, 150 mM NaCl, 0.5 mM TCEP) or crystallization buffer (20 mM HEPES pH 7.8, 500 mM NaCl, 0.5 mM TCEP). Fractions were pooled, concentrated to designated concentration for experiments or stored at −80 °C. KIF4A_1192-1232 was purified similarly except that it was heated at 80°C for 10 min and centrifuged (15,000 xg, 10 min, 4°C) prior to SEC purification (SEC buffer: 20 mM HEPES pH 7.8, 500 mM NaCl, 0.5 mM TCEP).

Following expression of human ncOGT (full length, 1–1046) in E.coli, cells were resuspended in a buffer containing 25 mM imidazole, 10 % glycerol, 250 mM NaCl, and 25 mM Hepes, pH 7.5, 5 mM β-mercaptoethanol. DNaseI and an EDTA-free protease inhibitor tablet (Sigma) were also added to the lysis buffer. Following lysis by sonication and French press, the protein was purified by nickel affinity chromatography and eluted in the same buffer with 250 mM instead of 25 mM imidazole. Fractions containing pure protein were then dialyzed in 25 mM Hepes, pH 7.5, 40 mM NaCl, 0.5 mM EDTA, and 5 mM β-mercaptoethanol and then loaded onto a 5 ml HiTrap Q-XL column (Cytiva) and purified at 4 °C using a gradient from 0.05 to 1.0 M NaCl. Although the protein appeared pure after this anion exchange step, we further purified the protein using a HiLoad Superdex 200 HR16/600 size exclusion column using a buffer containing 40 mM KPO₄, pH 7.5, 125 mM NaCl, 0.5 mM EDTA, 0.5 mM benzamidine, and 5 mM β-mercaptoethanol to ensure that no contaminating proteases remained.

Confluent MA104-GCaMP6s and MA104-GCaMP6s-IP₃R-TKO cells were inoculated with SA11-mRuby (MOI 10) and then harvested in RIPA buffer at 2, 4, 6, 8, and 10 hpi (21 (link)). The RIPA buffer composition was 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, and 1 cOmplete EDTA-free protease inhibitor tablet (Sigma-Aldrich). Samples were boiled for 10 min at 100°C in SDS-PAGE sample buffer and separated on Tris-glycine 4%–20% SDS-PAGE gels (BioRad) and blotted to nitrocellulose, which was blocked with 10% non-fat dry milk in PBS (BLOTTO). Primary antibodies were diluted in 0.5% BLOTTO and were rabbit anti-rotavirus [strain Alabama, (1:1,000)] (21 (link)), rabbit antisera to SA11 NSP4-aa120-146 (1:2,000) (25 (link)), and mouse anti-GAPDH (1:2,000). Alkaline phosphatase-conjugated secondary antibodies were used at a dilution of 1:2,000 in 0.5% BLOTTO. Membranes were incubated with primary antibodies overnight and incubated with secondary antibodies for approximately 2 h. After washing off secondary antibodies in 0.5% BLOTTO, membranes were developed using alkaline phosphatase detection solution (50 mM Tris, 3 mM MgCl₂, 0.1 mg/mL p-nitro blue tetrazolium chloride, and 0.05 mg/mL 5-bromo-4-chloro-3-indolyl phosphate).