Protease inhibitor cocktail set 5

Three 15-cm dishes each of hTERT-RPE1-mGFP (control) and hTERT-RPE1-mGFP-WDR34 cells were serum-starved for 24 h at confluence to induce ciliogenesis. Cells were briefly washed twice with 20 ml of ice-cold PBS per dish, and the PBS was drained off completely by leaning the dishes at ∼60° for ∼10 s. 500 µl of ice-cold lysis buffer 1 [10 mM Tris-HCl pH 7.4, 50 mM NaCl, 0.5 mM EDTA, 1.0% IGEPAL (CA-630, Sigma)], containing freshly added 1 mM phenylmethane sulfonylfluoride (PMSF) and 1× EDTA-free Protease Inhibitor Cocktail Set V (539137, Millipore, UK), was added to the cells, and the cells were scraped off the dish into 2-ml tubes. Cells were lysed by incubation with the buffer on a rotor at 4°C for 30 min. Lysates were centrifuged at 20,000 g at 4°C for 10 min, and supernatants were transferred into pre-cooled 2-ml Eppendorf tubes.

HEK293T cells (ATCC #CRL-3216) were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine at 37 °C with 5% CO₂. The cells were transfected with the various expression vectors using an established calcium phosphate method^{35 (link)}. To measure the half-life of Flag-tagged Rb mutants, transfected cells were metabolically labeled with [³⁵S] methionine/cysteine for 20 h, washed twice with phosphate-buffered saline (PBS), and chased for the indicated times in regular medium. Cells were harvested, washed with PBS, and lysed with buffer A (20 mM Tris-HCl [pH 7.4], 0.2% [v/v] NP-40, 150 mM NaCl, and 1 mM DTT) containing protease inhibitor cocktail set V (Millipore #539137). The extract was clarified by centrifugation at 20,000 × g for 10 min at 4 °C. PD150606 (Sigma #D5946), MLN4924 (Sigma #505477), carfilzomib (UBPBio #F1300), bortezomib (LC Laboratories #B-1408), MG132 (Enzo #BML-PI102), CIP (New England Biolabs, #M0290), anti-Flag (1:1,000; Sigma #F1804), anti-actin (1:1,000; Sigma #A2066), anti-16E7 (1:1,000; Santa Cruz #sc-51951), and InstantBlue (Expedeon #ISB1L) were purchased from the indicated manufactures. Quantification of bands was performed using ImageJ (National Institutes of Health, version 1.51).

The pneumococcal cells grown to the mid-log phase were resuspended in PBS. TIGR4 strains (3–11 × 10³ CFUs/well) with or without rPfbA (0, 10, or 100 nM) were combined with human neutrophils or neutrophil like-differentiated HL-60 cells (2 × 10⁵ cells/well), and R6 strains (1.4–2.0 × 10² CFUs/well) were combined with human neutrophils (1 × 10⁵ cells/well). The mixture was incubated at 37°C in 5% CO₂ for 1, 2, and 3 h. Viable cell counts were determined by plating diluted samples onto TS blood agar. The growth index was calculated as the number of CFUs at the specified time point/number of CFUs in the initial inoculum. Bacterial phagocytosis was blocked by addition of cytochalasin D (20 μM), and pneumococcal killing was blocked by protease inhibitor cocktail set V (Merck, Darmstat, Germany; 500 μM AEBSF, 150 nM Aprotinin, 1 μM E-64, and 1 μM leupeptin hemisulfate, EDTA-free) at 1 h before incubation. To determine whether TLR2 and TLR4 signaling affect pneumococcal survival, 100 μM TIRAP (TLR2 and TLR4) inhibitor peptide or control peptide (Novus Biologicals) were added to neutrophils at 1 h before incubation.

Birinapant and LCL-161 were purchased from MedChemExpress. zVAD-fmk was purchased from Bachem. Necrostatin-1 was obtained from Tocris Bioscience. MRT67307, TPCA-1 [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide and Protease Inhibitor Cocktail Set V were purchased from EMD Millipore.

Cells were washed with 1xPBS and frozen at −80 °C as a pellet. Cells were lysed on ice in RIPA buffer supplemented with protease inhibitor cocktail, set V (EMD Millipore). Protein concentration was measured with Pierce BCA protein assay. Equal amounts of protein were loaded onto 4–12% polyacrylamide gel (Bio-Rad Laboratories) and electrophoresed at constant power of 45 watts, for 40 min. Proteins were transferred from gels to PVDF membranes (Bio-Rad Laboratories) per manufacturer protocols. To intensify signals, PVDF membranes after protein transfer, were air dried at room temperature and then rehydrated with 100% methanol and 1×PBS with 0.05% Tween 20. Membranes were blocked for 20–30 min at room temperature with 5% non-fat milk prepared in 1×PBS, 0.05% Tween 20. Primary and secondary antibodies (Supplementary Materials) were prepared in 5% non-fat milk, 1×PBS, 0.05% Tween-20. Peroxidase activity was measured using HyGlo (Denville scientific) detected on a ChemDoc MP Imaging system (Bio-Rad).

Samples were lysed for 10 min on ice in lysis buffer containing 20 mM Tris-HCl pH 8.0, 137 mM NaCl, 10 mM EDTA, 100 mM NaF, 1% (v/v) Nonidet P-40, 10 mM Na₃VO₄, 2 mM PMSF, 1% (v/v) protease inhibitor cocktail set V (Merck Millipore) and 1% (v/v) phosphatase inhibitor cocktail set IV (Merck Millipore). Total protein concentration of lysates was quantified using a DC Protein Assay (BioRad) and 100 μg of each sample labelled using TMTsixplex reagents according to manufacturer’s instructions (Thermo Scientific). The labelled samples were pooled and then subjected to phospho-peptide enrichment using a TiO₂-based enrichment kit, according to the manufacturer’s instructions (Pierce). The phospho-enriched sample was then analysed by RP nano-LC MSMS using an LTQ-Orbitrap Velos mass spectrometer.