Millipore immobilon p pvdf membrane

Cells were collected in Cell Extraction Buffer (Invitrogen, FNN0011) with protease inhibitor (Roche, 04693116001) and PMSF (Sigma, P7626). Proteins were separated on 10% or 12% SDS-PAGE gels before transfer to Millipore Immobilon-P PVDF Membrane (Merck Millipore, IPVH00010). Membranes were probed with pAKT(S473) (Cell Signaling, 3787, 1:3,000), pan-AKT (Cell Signaling, 4691, 1:1,000), dpERK1/2 (Cell Signaling, 4370, 1:300), ERK1/2 (Cell Signaling, 4695, 1:300), SPRY2 (Abcam, Ab50317, 1:300), SOX2 (Abcam, Ab97959, 1:3,000), Histone H3 (Abcam, Ab39655, 1:10,000), and EGFR (Millipore, 04-290, 1:300). Detection was performed with horseradish peroxidase-conjugated secondaries (Abcam, ab97051, ab97023, 1:10,000) and enhanced chemiluminescence (Thermo Scientific, PI-32109). Quantitation is based on analysis of protein from three biological replicates separated on the same polyacrylamide gel. Band intensity was analyzed in Fiji normalized to loading control.

Cells were collected in Cell Extraction Buffer (Invitrogen, FNN0011) with protease inhibitor (Roche 04693116001) and PMSF (Sigma, P7626). Proteins were separated on 10% or 12% SDS-PAGE gels before being transfer onto Millipore Immobilon-P PVDF Membrane (Merck Millipore, IPVH00010). Primary antibodies are listed in Table S1. Detection with HRP-conjugated secondaries (Abcam, 1:10,000) and enhanced chemiluminescense (Thermo Scientific, PI-32109) was carried out. Quantitation is based on protein from three biological replicates separated on the same polyacrylamide gel. Band intensity was analysed in Fiji normalised to the loading control.

EVs were lysed with RIPA buffer (Abcam, Inc., Cambridge, UK) containing 60 mg/mL dithiothreitol and SIGMAFAST™ Protease Inhibitor tablets (Sigma-Aldrich, St. Louis, MO, USA) and the protein amounts were quantitated using a Bradford assay. Proteins (20 µg) were separated by 12.5% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore Immobilon®-P PVDF Membrane, Merck KGaA, Darmstadt, Germany). After blocking with 5% skim milk, the membrane was incubated with a 1:1000 primary antibody as follows: anti-Alix (#2171; Cell Signaling, Danvers, MA, USA), anti-Hsp70 (ab79852; Abcam plc, Cambridge, UK), anti-Tsg101 (ab125011; Abcam plc.), anti-n-Myc (ab24193; Abcam plc.), or anti-calnexin (#40090; Cell Signaling), at 4 °C overnight. The probed membrane was washed and incubated with 1:1000 anti-rabbit (P0448; Dako, Glostrup, Denmark) or 1:2000 anti-mouse (P0447; Dako) as an appropriate at room temperature for 1 h. The detected protein bands were enhanced by SuperSignal West Pico chemiluminescence substrate (Thermo Scientific) and captured by G:BOX Chemi XRQ (SYNGENE, Cambridge, CB4 1TF, UK).