G 6983

PD0325901 (Chemscence, Monmouth Junction, NJ), a MEK inhibitor; Gö 6983 (Abcam, Cambridge, UK), a protein kinase c (PKC) inhibitor; MK-2206 (Selleck, Houston, TX), a protein kinase b (AKT) inhibitor; Losmapimod (Selleck), a p38α/β mitogen-activated protein kinase (p38 MAPK) inhibitor; SP600125 (Selleck), a c-jun N-terminal kinase (JNK) inhibitor; Saracatinib (Selleck), a proto-oncogene tyrosine-protein kinase Src (SRC) inhibitor and JAK Inhibitor I (Santa Cruz Biotechnology, Dallas, TX), a janus kinase (JAK) inhibitor, were dissolved in dimethyl sulfoxide (DMSO) (Sigma, ST. Louis, MO) and added to the culture medium. Pure DMSO was used as the negative control.

Deferoxamine, Forskolin, 8-bromoadenosine 3′,5′-cyclic monophosphate (cAMP), phorbol 12-myristate 13-acetate (PMA), COCl₂, thapsigargin (Tg), and staurosporine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Gö6983 was obtained from Abcam (Cambridge, UK), and mithramycin-A was purchased from Cayman Chemicals (Ann Arbor, MI, USA). Expression plasmids of wild type Sp1 and functionally inactive mutant Sp1, as previously reported [86 (link)], were purchased from Addgene, Watertown, MA, USA, (#12098 and #12097, respectively) and sub-cloned to pcDNA3 vector. Catalytically active mutant isoforms plasmids of PKC family were kindly donated by Professor, Jae-Won Soh (Inha University, Incheon, Korea). Details of the PKC mutant isoforms were previously published [23 (link)].

R848 (TLR7/8 agonist; InvivoGen) was reconstituted in sterile water at a concentration of 3 mM. TDB (Mincle agonist; Avanti Polar Lipids) was reconstituted in dimethyl sulfoxide (DMSO; Sigma-Aldrich) at a concentration of 20 mg/mL, heated at 60 °C for 30 s, then brought to 2 mg/mL using sterile Dulbecco’s PBS (DPBS) without Ca₂+, Mg₂+, and phenol red (Life Technologies) and heated again at 60 °C for an additional 15 min. 3 M052 (TLR7/8 agonist) was a kind gift from Dr. Mark Tomai, 3M Drug Delivery Systems.
PKC-δinhibitor peptide (SFNSYELGSL; Anaspec Inc.), PKC inhibitor (Gö 6983; Abcam) and Clathrin inhibitor (Pitstop®1, and corresponding negative control; Abcam) was reconstituted in DMSO at 10 mM and used at 10 μM concentration in cell culture.
All agonists and inhibitors were verified to be endotoxin-free (<1 EU/ml), as measured by Limulusamebocyte lysate (LAL) assay per the manufacturer’s instructions (Charles River). Sterile DPBS or DMSO were included in experiments at appropriate concentrations as a negative control when indicated.