Anti pyk2

The following 21-nucleotide duplex siRNAs against human CXCR7 (siCXCR7-1, siCXCR7-2), LCP1 (siLCP1-1, siLCP1-2) as well as a control (siControl), were synthesized: siCXCR7-1, 5′-ccuucauuuacauuuucaudTdT-3′, 5′-augaaaauguaaaugaaggdTdT-3′; siCXCR7-2, 5′-ccuuauaa-auguauuugaadTdT-3′, 5′-uucaaauacauuuauaaggdTdT-3′; siLCP1-1, 5′-gcuuugaugaguuuaucaadTdT-3′, 5′-uugauaaacucaucaaagcdTdT-3′; siLCP1-2, 5′-gaacaaucaacaaaaagaadTdT-3′, 5′-uucuuuuuguugauuguucdTdT-3′; siControl, 5′-cguacgcggaauacaacgadTdT-3′, 5′-ucguuguauuccgcguacgdTdT-3′. Used antibodies were as follows: anti-CXCR4 (Abcam, USA), anti-CXCR7 (Abcam, USA), anti-LCP1 (GeneTex, USA), anti-GLI1 (Novus), anti-GLI2 (Santa Cruz Biotechnology), anti-GAPDH (Santa Cruz Biotechnology, USA), anti-ERK1/2, anti-phosphorylated ERK1/2, anti-PYK2 and anti-phosphorylated PYK2 (Cell Signaling).

Recombinant human VCAM-1/CD106 Protein and Recombinant Human/Rhesus Macaque/Feline CXCL12/SDF1α were from R&D Systems (Minneapolis, MN, USA). Human fibronectin, doxorubicin and FAK inhibitor PF-228 were purchased from Millipore Sigma (Oakville, ON, Canada). The PYK2 inhibitor VS-6063 was purchased from Cayman Chemicals (Ann Arbor, MI, USA). Anti-phospho-PYK2 (Tyr-402) and anti-PYK2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The fluorescein isothiocyanate (FITC)-conjugated Annexin V, Phycoerythrin (PE)-conjugated anti-human α4 integrin, Alexa 647-conjugated anti-human α5 integrin antibodies, FITC-conjugated anti-human CD3 and PE-conjugated anti-mouse IgG were purchased from BD Biosciences (San Jose, CA, USA). Anti-human β1 integrin (4B4) was purchased from Beckman Coulter Life Sciences (Mississauga, ON, Canada).

PMA (phorbol 12-myristate 13-acetate), ATP, nigericin, CCCP, PP2, dAdT, and MG132 were purchased from Sigma. MitoSox, H₂-DCFDA, TMRE, MitoTracker Green FM, and Hoechst were purchased from Life Technologies. MitoTEMPO was purchased from Enzo Life Sciences. Hydrocotarnine was purchased from Enamine. Anti-Pyk2, anti-AIM2, and anti-p-Pyk2 were purchased from Cell Signaling. Anti-Cbl, anti-ASC, anti-caspase-1, anti-IL-1β, anti-phosphotyrosine, and anti-GAPDH were purchased from Santa Cruz. Anti-Ly6G, anti-CD45, and anti-CD11b were purchased from BD Bioscience. Anti-NLRP3 and anti-F4/80 were purchased from BioLegend and eBioscience, respectively. Plasmids encoding mutants CBL (Y371D) and CBL (Y371F) were generated by ligating amplified DNA fragments into the NheI/PmeI-treated pLKO_AS2.neo vector (RNAi Core, Taiwan). The mutant CBL was constructed using a QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies), and the following primers (forward and reverse, respectively): for Cbl Y371D, 5′-CAGGAACAATATGAATTAGACTGTGAGATGGGCTCCAC-3′ and 5′-GTGGAGCCCATCTCACAGTCTAATTCATATTGTTCCTG-3′; and for Cbl Y371F, 5′-CAGGAACAATATGAATTATTCTGTGAGATGGGCTCCAC-3′ and 5′-GTGGAGCCCATCTCACAGAATAATTCATATTGTTCCTG-3′.

Alpha-minimum essential medium (α-MEM) and fetal bovine serum (FBS) were purchased from Life Technologies (Carlsbad, CA, USA). Recombinant human M-CSF was purchased from R&D Systems (Minneapolis, MN, USA), and GST-RANKL was purchased from Oriental Yeast Co., Ltd (Shiga, Japan). Anti-trimethyl-histone H3 lysine 4 and anti-trimethyl-histone H3 lysine 27 were from Active Motif (rabbit polyclonal antibody, 39159, Carlsbad, CA, USA) and Millipore (rabbit polyclonal antibody, 07–449, Billerica, MA, USA), respectively. Anti-NFATc1 antibody detects all DNA binding domain-containing NFATc1 splicing isoforms, but not the closely related NFATc2 [27 (link)]. Anti-FAK, anti-Pyk2 and anti-Src antibodies were from Cell Signaling Technology (rabbit polyclonal antibody; Beverly, MA, USA).

Cells were lysed with a 1% Triton X-containing lysis buffer, as described previously [22 (link)]. Lysates were boiled with sodium dodecyl sulfate (SDS) sample buffer for 5 min at 95 °C [22 (link)]. Samples were separated on 7.5, 10, 12 and 15% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% bovine serum albumin (BSA), the membranes were incubated with primary antibodies. The antibodies used were anti-Plectin-1 (#12254), anti-Src pY416 (#2101), anti-Pyk2 (#3292), anti-Pyk2 pY402 (#3291), anti-p38 MAPK (#8690), anti-phospho-p38 MAPK (Thr180/Tyr182) (#9211), anti-p44/42 MAPK (Erk1/2) Antibody (#9102), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#4370) (Cell Signaling Technology, CST, Danvers, MA), anti-Src (ab-1) (Merck, Darmstadt, Germany), anti-DDDDK-tag (Flag-tag, Fla-1), anti-GAPDH mAb-horseradish peroxidase-conjugated (HRP)-DirecT (Medical & Biological Laboratories, MBL, Tokyo, Japan), and β-actin (A2228, Sigma Aldrich Chemicals, St. Louis, MO). The membranes were then incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies. Finally, the blots were imaged using a LAS4000 (Fujifilm Wako) with Immobilon ECL Ultra Western HRP Substrate (Merck).

Anti-Pyk2 was obtained from Cell Signaling Technology (3292); anti-FAK (clone 77; 610088) was obtained from BD Biosciences. Additional anti-FLAG (clone M2; F3165) and an anti-β actin (clone AC-15; A5441) antibody were both obtained from Sigma-Aldrich (St. Louis, MO, USA). Secondary antibodies (goat anti mouse 680LT and goat anti rabbit 800CW) were obtained from Li-COR Biosciences.
Mice were rapidly decapitated, and brains were rapidly frozen at −80 °C for later dissection. Alternatively, transfected HEK293T or snap-frozen lysates were washed in ice-cold PBS and lysed in Triton lysis buffer (1% Triton, 10% glycerol, 120 mM NaCl, 25 mM HEPES, 1 mM EDTA, 0.75 mM MgCl_2, 2 mM NaF, 1 mM Sodium orthovanadate, and protease inhibitors). The total protein concentration was determined using a DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA), and equal amounts were loaded on SDS-PAGE, transferred to a nitrocellulose membrane, and blocked in an Odyssey blocking buffer. Membranes were incubated with primary and secondary antibodies and imaged using the Odyssey CLx imaging system (LI-COR Biosciences, Lincoln, NE, USA). The samples were quantified using an Odyssey CLx imaging system (LI-COR Biosciences), and the values were normalized to the loading control and presented as a fold change from the control mean.