Anti β actin clone ac 74

Total cellular proteins were separated on 4–12% NuPAGE Bis-Tris gel (Life Technologies) before transferring to PVDF membrane (Pall Australia, VIC, Australia). The membrane was blocked, incubated with anti-Hyaluronic acid mAb (LS-C315053) (Sapphire Bioscience, NSW, Australia). Anti-β-actin (clone AC-74) (Sigma-Aldrich) was used as the internal control, followed by horseradish peroxidase-conjugated secondary antibody and subjected to enhanced chemiluminescence (Roche, VIC, Australia).

Twenty micrograms of total cellular and EV lysates were separated by SDS-PAGE before transferring to PVDF membrane (Pall Australia, VIC, Australia), as described in [4 (link)]. The membrane was blocked overnight with 5% skim milk in PBS and 0.05% Tween 20 and then incubated with anti-P-gp mAb (clone F4; Sigma-Aldrich), anti-Ezrin mAb (clone 3C12; Life Science), anti-Moesin mAb (clone 38/87; Sigma-Aldrich), anti-Radixin pAb (SAB2500859; Sigma-Aldrich) or anti-CD44 pAb (HPA005785; Sigma-Aldrich) for 1 h. Anti-β-actin (clone AC-74; Sigma-Aldrich) was used as the internal control, followed by 1 h incubation with anti-Mouse HRP secondary antibody (Promega, NSW, Australia) at a 1:10,000 dilution. Protein expression was visualised using the ECL (enhanced chemiluminescence) system (Roche Applied Science, Castle Hill, NSW, Australia). The membranes were imaged using the luminescent image analyser LAS-3000 (Fujifilms, Brookvale, NSW, Australia).

The following antibodies were used in this study: anti-Girdin (R&D Systems, Minneapolis, MN, #AF5345, for WB and IP), anti-4F2hc (H300, Santa Cruz Biotechnology, Santa Cruz, CA, for WB, clone MEM-108, Biolegend [#315602] for IP, and mouse monoclonal anti-4F2hc [clone HBJ 127] for IF), anti-glutathione S-transferase (GST) (Santa Cruz Biotechnology, #sc-459), anti-Myc (clone 9E10, Santa Cruz Biotechnology), anti-poly-histidine (clone HIS-1, Sigma, St. Louis, MO), anti-Flag (clone M2, Sigma), anti-β-actin (clone AC-74, Sigma), normal mouse IgG (Millipore, Milford, MA, #12–371), and normal sheep IgG (Millipore, #12–515). Antibodies to pS6K (Thr389) (#9205), S6K (#9202), S6 (#2217), pS6 (Ser240/244) (#2215), MAPK (#9102), pMAPK (Thr202/Tyr204) (#9106), Lamp1 (#9109), mTOR (#2983), and LC3B (#2775) were purchased from Cell Signaling Technology (Danvers, MA). Flag M2 affinity gel, adenosine 5′-triphosphate (ATP), and amino acids were purchased from Sigma. Phos-tag Acrylamide (Wako, Saitama, Japan) was used for the generation of the Phos-tag gel to analyze protein phosphorylation in cells.

Leukemic bone barrow cells from p19Arf^-/-Rag1^-/-, p19Arf^-/-Rag1^-/-Aid^+/- and p19Arf^-/-Rag1^-/-Aid^-/- mice, switch culture stimulated spleen cells from WT C57BL/6 mice (LPS (20 μg/ml) + IL4 (25 ng/ml) treatment for 4 days) or BaF3 cells (with and without expression of murine Jak3 V670A and after 4h of IL-3 depletion) were lysed in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0,5 % Sodiumdeoxycholate, 1 % NP-40 substitute, 0,1 % SDS) containing protease/phosphatase inhibitors (Roche Diagnostics). Immunoblotting was carried out using the following antibodies: anti-AID clone L7E7 1:1000 (Cell Signaling), anti-phospho-Stat5 D47E7 (Tyr694) 1:1000 (Cell Signaling), anti-Stat5 1:1000 (Cell Signaling) and anti-β-Actin clone AC-74 1:10000 (Sigma-Aldrich). Detection was carried out using anti-mouse horseradish peroxidase conjugates (Santa Cruz Biotechnology) with an ECL system (Thermo Scientific) (n = 3).

Cells and sEVs were lysed in RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM NaF, 0.1 mM sodium orthovanadate) supplemented with protease inhibitor cocktail (cat. no., 25955; Nacalai Tesque). Lysates and immunoprecipitates were denatured in Laemmli buffer containing DTT and 2-mercaptoethanol, respectively. The denaturation was performed for 2 h at 37 °C for the lysates of EVs and for 5 min at 100 °C for the other samples. After cooling, the proteins were separated by SDS-PAGE, transferred onto PVDF membranes, blocked with 5% milk or 2% BSA for 1 h and probed with following primary antibodies: anti-Alix (12422-1-AP; Proteintech), anti-β-actin (clone AC-74; SIGMA), anti-CD9 (ab92726; Abcam), anti-ephrin-A1 (AER-031; Alomone Labs), anti-EphA2 (C-20; Santa Cruz), anti-Erk (#9102; CST), anti-FLAG (PM020; MBL), anti-p21 (C-19; Santa Cruz), anti-phospho-Erk (E-4; Santa Cruz), anti-phospho-p53 (Ser15) (#9284; CST), anti-phosphotyrosine (clone 4G10; Millipore), anti-PTP1B (clone FG6-1G; Millipore) and anti-Rab35 (11329-2-AP; Proteintech). All primary antibodies were diluted to 1 μg ml⁻¹. After washing, membranes were incubated with secondary antibodies (GE Healthcare) (1:1,000) for 1 h at room temperature (RT) and visualized with enhanced chemiluminescence reagents. Uncropped gel images are provided in Supplementary Figs 6–12.