Typhoon gel scanner

Purified SHIP1, YopH, and c-Src were resolved on a 4 to 12% Mini-PROTEAN TGX precast gradient gel (Bio-Rad; catalog no.: 4561094) at 200 V for 30 min. Proteins resolved by SDS-PAGE were transferred to nitrocellulose membranes (Bio-Rad; catalog no.: 1704271) using cold 1× transfer buffer (40 ml 5× transfer buffer [Bio-Rad; catalog no.: 1704271], 40 ml ethanol, and 120 ml Milli-Q water). Protein transfer to nitrocellulose membrane was accomplished using the 7 min mixed molecular weight protocol on the Bio-Rad Trans-Blot Turbo Transfer System (Bio-Rad; catalog no.: 1704150). Blots were washed with Milli-Q water and blocked for 1 h with intercept blocking buffer (LI-COR; catalog no.: 927-70001) at 23 °C. The nitrocellulose membrane was then incubated with 1:250 Alexa Fluor 546–labeled phosphotyrosine primary antibody (PY20) (Santa Cruz Biotechnology; catalog no.: sc-508-AF546) diluted in intercept blocking buffer and rocked overnight at 4 °C in the dark. Before visualization, the nitrocellulose membrane was washed four times for 5 min with 1× PBS plus 0.1% Tween-20. The Western blot was visualized using an Amersham Typhoon gel scanner. Data are shown in Fig. S4D.

Samples (55 μL) were prepared for SDS-PAGE by adding 20 μL NuPAGE (LDS) buffer (Thermo Scientific, Hertfordshire, UK), and 5 μL of either 0.5 M dithiothreitol (DTT) (reduced samples) or ultrapure water (non-reduced samples). Reduced samples were heated to 100 °C for 5 min and cooled to ambient temperature prior to loading to 4–12% Bis–Tris gels in a NuPAGE system (Invitrogen, Thermo-Fisher Scientific, Paisley, UK). Mark 12 Unstained Standard (Invitrogen) was used as molecular weight markers for protein-stained gels and SeeBlue pre-stained markers (Invitrogen, Loughborough, UK) were used for gels to be electroblotted. Proteins were separated according to the manufacturer’s instructions using 200 V, 350 mA and 100 W for 35 min. Gels were fixed in 50% (v/v) methanol, 10% (v/v) acetic acid and after 1 h rinsed three times for 5 min each in deionised water before staining with Coomassie G-250 stain (SimplyBlue, Invitrogen). The gel was de-stained by rinsing with MilliQ water and imaged using a Typhoon gel Scanner (Amersham, UK).

Samples were prepared as described before (Kocaoglu et al., 2015 (link)) with slight modifications. Briefly, 4 mL of cells were grown in C+Y pH 6.8 until OD 0.15 and harvested by centrifugation (16,000 × g for 2 min at 4°C). Cell pellets were washed in 1 mL PBS, pH 7.4. Cells were pelleted and resuspended in 50 μL PBS with or without the indicated concentration of ATM or CLA. After 30 min of incubation at room temperature, cells were pelleted, washed in 1 mL PBS, and resuspended in 50 μL PBS containing 5 μg/ml Bocillin-FL. After 10 min of incubation at room temperature, cells were washed again in 1 mL PBS. Next, cells were sonicated on ice (power 30%, three cycles of 10 s interval with a 10 s cooling time on ice (Sonoplus, Bandelin). Then samples were centrifuged at max speed for 15 min at 4°C and pellets were resuspended in 100 μL cold PBS. The protein concentration was adjusted to 2 mg/ml as determined by Bradford by diluting with PBS. 5x SDS-PAGE loading buffer was added to each sample and heated 10 minutes at 95°C. Proteins were separated by gel electrophoresis (10% acrylamide) for 2.5 h at 180 V, 400 mA, and 60 W. The gel was scanned using a Typhoon gel scanner (Amersham Biosciences, Pittsburgh, PA) with a 526-nm short-pass filter at a 25-μm resolution.