Flag tag (flag)

Tumors were mechanically dissociated into single cell suspensions as previously described [49 (link)]. Cell suspensions were stained with conjugated antibodies (Biolegend, BD or eBiosciences) and Zombie aqua (Sigma). Following strategy was used to identify cells of interest:
Fluorochromes employed were the following: eGFP, Bv421, Bv605, Bv785, PE, PerCP, PC7, APC, A700, AC7.
Single EV analysis (S2B, S2C Fig) was performed as previously described [50 (link)]. Briefly, a 10-fold dilution of stock EV preparations in 0.2um filtered PBS was further serially diluted 2-fold to generate a 4-point dilution series. EV dilution series was stained with the following antibodies: CD63, CD81 and Flag, all from Biolegend and diluted to 0.1 ug/ml, without washing. Control unstained EV dilution series was also prepared. For each sample, we used a SSC trigger threshold value of 200 and the window extension at 3msec. EV staining samples were performed and analyzed in triplicate. After acquisition of 60ul (out of 200ul total), Triton-X100 was added to a final concentration of 0.5% (v/v) in each well and the plate was acquired again to confirm staining of EVs.

CP13, PHF1 and MC1 antibodies were kind gifts from P. Davies (Albert Einstein College of Medicine). Other antibodies were purchased from commercial sources as follows: AT8 (Pierce) and AT100 (Innogenetics), total Tau (DAKO), Tau1 (Millipore), PTEN (Cell Signaling), LAMP1 (Millipore), LC3 (Novus Biological and Sigma), Akt (Cell Signaling) and pAkt (Cell Signaling), P70S6K (Cell Signaling) and pP70S6K (Cell Signaling), ULK1 (Cell Signaling) and pULK1 (Cell Signaling), Y188 (Epitomics), 6E10 (Covance), NeuN (Chemicon), GFAP (DAKO), Iba1 (Waco), GAPDH (Sigma), FLAG (Biolegend), human TFEB (Cell Signaling), mouse and human TFEB (Abcam and Abmart), and γ-tubulin (Sigma).