Ha probe f 7

The following reagents and antibodies were purchased from commercial sources: inhibitor cocktail (Trichostatin A (TSA, T8552, Sigma), protease inhibitor cocktail (P8340, Sigma), phosphatase inhibitor cocktail (P0044, Sigma)), fedratinib (S2736, Selleckchem), universal nuclease (88700, Thermo Fisher), Bradford assay (23200, Thermo Fisher), dithiothreitol (DTT; DTT100, Goldbio), enzyme-linked chemiluminescence (ECL) plus (32132, Thermo Fisher), SYBR Green PCR Master Mix (4472908, Applied Biosystems), streptavidin agarose (20359, Thermo Fisher), Protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology), anti-Flag agarose gel (A2220, Sigma) and anti-HA affinity gel (E6779, Sigma). Antibodies were as follows: STAT3 (9139, CST), phospho-STAT3 (Tyr705) (ab76315, Abcam), β-actin (C4) HRP (SC-47778, Santa Cruz), Na/K-ATPase (SC-21712, Santa Cruz), histone H3 (4499S, CST), Flag HRP (A8592, Millipore), HA-probe (Y-11) (SC805, Santa Cruz), HA-probe (F-7) (SC7392, Santa Cruz), DHHC7 (ab138210, Abcam), DHHC7 (R12–3691, Assay Biotechnology), Alexa Fluor 350 goat anti-rabbit IgG (A-11046, Invitrogen), Alexa Fluor 594 goat anti-mouse IgG (8890S, CST), mouse CD4 PerCP-Cy5.5 (560767, BD Pharmingen), mouse IL-17A PE (560767, BD Pharmingen), anti-mouse IgG HRP (7076S, CST) and anti-rabbit IgG HRP (7074S, CST).

HEK293T cells were plated onto poly-L-Lysine-coated coverslips and then transfected with HA-tagged Thpok or Thpok mutants. 48 hours post transfection, cells were fixed with 2% paraformaldehyde for 30 min at room temperature. After three washes with PBS, the coverslips were incubated in blocking buffer (PBS, 3% BSA, 0.5% Triton X-100, 10% newborn calf serum) at room temperature for 1 hour followed by three washes with binding buffer (PBS, 3% BSA, 0.5% Triton X-100). Samples then were incubated with 2 μg/ml HA-probe (F-7, Santa Cruz Biotechnology) at room temperature for 1 hour. After three washes with binding buffer, samples were incubated with 10 μg/ml goat anti-mouse Alexa Fluor 555 (Thermo Fisher) at room temperature for 1 hour. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (Life Technologies). Samples were mounted on microscope slides using Mowiol (Sigma) and examined on Axioplan 2 imaging.