Phosphatase inhibitors cocktail

Cells were rinsed twice with PBS, resuspended in ice-cold lysis buffer (0.1% deoxycholic acid, 1% Triton X-100 in PBS) containing a protease (Roche, Basel, Switzerland) and phosphatase inhibitors cocktail (Sigma Aldrich, Saint-Louis, MO, USA) and cleared by centrifugation. Protein concentrations were quantified using the BCA protein assay reagents (Pierce, Waltham, MA, USA). The extracts (40 µg/lane) were resolved in 10% SDS-PAGE and transferred to a PVDF membrane (Macherey-Nagel, Düren, Germany). Protein bands were visualized using an enhanced chemiluminescence kit (GE Healthcare Bio-Sciences, Chicago, Illinois).

Cells grown in confluent monolayers were rinsed twice with PBS, resuspended in ice-cold lysis buffer (1% Triton X-100; 50 mM Tris pH7.5; 150 mM NaCl; 0.5% Nonidet P-40) containing protease (Roche) and phosphatase inhibitors cocktail (Sigma Aldrich), disrupted by sonication and cleared by centrifugation. Protein concentrations were quantified using the BCA protein assay reagents (Pierce). The extracts (100 μg/lane) were resolved in 8% SDS-PAGE and transferred to a PVDF membrane (Macherey-Nagel). Protein bands were visualised using an enhanced chemiluminescence kit (GE Healthcare Bio-Sciences) and quantified in ImageJ software (using Analyze-Gels). All results were normalised to actin.

For immunoprecipitation experiments, the cells were lysed with immunoprecipitation lysis buffer (Thermo Fisher Scientific) containing protease inhibitors and phosphatase inhibitors cocktail (Sigma-Aldrich). Cell lysates were then incubated with anti-SLIT2 antibody and protein A/G beads (Thermo Fisher Scientific) at 4°C for 6 h. After being washed with immunoprecipitation lysis buffer, the beads were boiled for 5 min with 2× SDS loading buffer. Eluted proteins were used for Western blotting analysis.

For western blotting (WB), cells grown in confluent monolayers were rinsed twice with cold PBS, resuspended in ice-cold lysis buffer (1% Triton X-100; 50 mM Tris pH 7,5; 150 mM NaCl; 0,5% Nonidet P-40) containing protease (Roche) and phosphatase inhibitors cocktail (Sigma Aldrich), disrupted by sonication and cleared by centrifugation. Protein concentrations were quantified using the BCA protein assay reagents (Pierce). Protein extracts (100 μg/lane) were resolved in 8% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Macherey-Nagel). The total level of CA IX protein was detected by HRP-conjugated M75 antibody, and HIF-1α and actin were detected using purified primary antibodies and the appropriate HRP-conjugated secondary antibodies as described in the section Antibodies. Protein bands were visualized using an enhanced chemiluminescence kit (GE Healthcare Bio-Sciences).

Tissues were ground three times in a mini bead beater in presence of lysis buffer (50 mM hydroxyethyl piperazineethanesulfonic acid (HEPES), 150 mM sodium chloride, 10 mM EDTA, 10 mM sodium pyrophosphate tetrabasic anhydrous, 25 mM β-glycerophosphate, 100 mM sodium fluoride, 10% glycerol anhydrous) supplemented with phosphatase inhibitors cocktail (Sigma Aldrich). Successive centrifugations were done to collect supernatant. Protein quantification was performed using a BCA protein assay kit (Pierce, Thermo Scientific). Bovine serum albumin (BSA) standard curve and sample preparation and analysis were realized according to manufacturer’s instructions. For protein immunoblotting, 20–30 micrograms of proteins were loaded for separation by SDS-PAGE electrophoresis and transfer on PVDF membranes. Membranes were then immunoblotted with the appropriate antibody to detect glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine 473 phosphorylated AKT, total AKT. Antibody binding was detected using HRP-conjugated secondary antibodies and ECL western blotting substrate (Thermo Scientific). Immunoblots were visualized by chemiluminescence imaging system (MF ChemiBIS 2020, DNR bio imaging systems, Jerusalem, Israel) and quantified using MultiGauge V3.2 software.

Cells were harvested by trypsinization and lysed in TEB250 lysis buffer (50 mM HEPES pH 7.4, 250 mM NaCl, 2 mM MgCl₂, 5 mM EGTA pH 8, 1 mM DTT, 0.5% Triton X-100, 10% glycerol, protease inhibitor cocktail (Roche), phosphatase inhibitors cocktail (PhosSTOP, Sigma), 50 μM PARPi, 100 μM PARG inhibitor and benzonase (Sigma; 1:1,000)) for 45 min on ice, followed by centrifugation (15 min at 16,000g). For immunoprecipitation, 0.5 mg or more of the protein lysate was incubated with antibody to mono/poly-ADP-ribose antibody (Cell Signalling; Supplementary Table 1) for 2 h and then with Protein G Dynabeads (Invitrogen) for 2 h. The beads were then washed (3×) with lysis buffer, resuspended in sample buffer and heated for 5 min at 90 °C and subjected to SDS–PAGE. The proteins were transferred to nitrocellulose and immunoblotted with ADP-ribose-binding reagent (Millipore; Supplementary Table 1).