Anti tubulin cy3

LM3 cells were seeded and grown 80% confluent until treatment with 80 µM hemin or vehicle. Immunofluorescence was performed as previously described^{23 (link)}. Rabbit anti-talin (1:100, Sigma), anti-tubulin-Cy3 (1:300, Sigma) and anti-GEF-H1 (1:500, Abcam) primary antibodies were used. For actin filaments, we used staining with Phalloidin-TRITC (1:300; Invitrogen) for 1 h at room temperature. Secondary Alexa 546 and 488 fluoro-conjugated antibodies (Molecular Probes) were used. DAPI was used to stain the nucleus. The coverslips were mounted on slides with mowiol mounting medium and we obtained superresolution and confocal z-stacks images in Zeiss LSM900 microscope with an Airyscan 2 module. Orthogonal x/y, x/z or y/z projection views were performed with Zeiss ZEN Software. Image intensity and protein distribution were analysed with Image J free software.

Using a CD4+ T cell negative selection kit (StemCell Technologies Inc.), CD4+ T cells were purified from lymph nodes and spleens of 6–8 week old C57BL/6 mice, which were bred in the animal care facility in POSTECH Biotech Center (PBC). All experiments involving mice were approved by the Institutional Animal Care and Use Committee in PBC. Anti-CD3 (clone: 2C11) was purchased in a large scale from BioXCell and custom labeled with N-hydroxysuccinimide (NHS)-activated forms of fluorescein (Pierce), Alexa Fluor 555 (Invitrogen), and Alexa Fluor 647 (Invitrogen) according to the instructions from the vendors. Anti-CD28 (clone: 37.51) was purchased from BioXCell, anti-TCRβ-FITC (clone: H57-597), anti-T-bet (clone: eBio4B10), and isotype control IgG were purchased from eBioscience, anti-tubulin-Cy3 (clone: TUB 2.1) was purchased from Sigma-Aldrich, anti-PKCζ was purchased from Abcam, and ICAM-1/Fc was purchased from R&D systems.