Mouse anti rab11

The following primary antibodies
were used for immunofluorescence: mouse anti-LAMP1 (1/10.000) (H4A3
clone, Developmental Studies Hybridoma Bank), mouse anti-EEA1 (1/100)
(Transduction Laboratories, 610456), mouse anti-RAB11 (1/100) (BD
Transduction, 610,657). Donkey antimouse-AF647 (1/300) (Invitrogen
A32787) antibody was used as a secondary antibody. DAPI (Sigma, D9542)
at 0.1 μg/mL and HCS Cell Mask Blue Stain (1/400) (Invitrogen,
H32720) were used to mark nuclei and cytoplasm. The following tagged
transferrin was used: Tf-AF647 (1/1000) (Molecular Probes, T23366)
and Tf-488 (1/1000) (Molecular Probes).
The following primary
antibodies were used for immunoblotting: mouse anti-CHC (abcam ab2731)
(1/500), goat anti-DNM2 (Santa Cruz, SC-6400) (1/1000), and rabbit
anti-GAPDH (Sigma G9545) (1/6000). Goat antimouse-HRP (Amersham, NA931)
(1/10000), rabbit anti-goat-HRP (Biodesign, W99119P) (1/10,000), and
donkey antirabbit-HRP (Amersham, NA9340) (1/10000) were used as secondary
antibodies.
To measure cytotoxicity, the Cytotox96 Non-Radioactive
cytotoxicity
assay kit (Promega) measuring the extracellular LDH has been used
after a 6 h incubation at a concentration of 6 nM.

T cells (Jurkat or SAg‐T blasts) alone or in conjugates with Raji cells were seeded onto coverslips precoated with poly‐L‐lysine (Sigma) prior to fixation with 4% PFA for 10 min at RT. After three washings with PBS, the coverslips were then incubated with NH₄Cl for 10 min to remove residual PFA. TCR and F‐actin were stained with anti‐CD3 Ab (clone UCHT1, Biolegend) and Phalloidin AF647 (ThermoFisher, A22287), respectively, followed by staining with secondary antibodies for 45 min at RT. Depending on the Abs, several protocols were used for intracellular staining. For mouse anti‐human Lck, permeabilisation with PBS supplemented with 0.05% saponin was performed for 15 min at RT, followed by incubation with the Ab diluted in the same buffer for 1 hr. For rabbit‐anti human‐GM130 (Abcam, ab52649) and mouse‐anti‐Rab11 (BD Bioscience, 610,657), permeabilisation was done with PBS supplemented with 0.1% Triton. Confocal microscopy was performed on a SP5 (Leica) or on an Opterra (Bruker) using either a 63× objective or a 100× objective (NA 1.4). Z‐stack acquisitions of optical sections were performed at 0.3 increments for analysis. Images were analysed using Fiji analysis software (Schindelin et al., 2012).