Phospho pyk2 tyr402

The human chondrocyte cell line C28/I2 was used^{68 (link)}. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Lonza) containing 10% FBS (Hyclone) and 1% penicillin/streptomycin (Lonza), and maintained at 37 °C and 5% CO₂ in a humidified incubator. Neon transfection system (Invitrogen) was used to transfect the biosensors into the cells. After transfection, two parts 3% agarose gel solution was mixed with one part 3 × DMEM containing transfected cells to produce the cell/gel construct with 2% agarose and 1 × DMEM. The mixture was injected into the μ-slide flow chamber (Ibidi) and cooled at room temperature for 30 min to allow gelation. The cell/gel construct was incubated in DMEM containing 0.5% FBS for 24 h before imaging 7 experiments. For Western blot analysis, cells were lysed in a radioimmunoprecipitation assay (RIPA) buffer. Isolated proteins were fractionated using 10% SDS gels and electro-transferred to Immobilon-P membranes. The membrane was incubated with primary antibodies, followed by incubation with secondary antibodies conjugated with horseradish peroxidase (Cell Signaling). The primary antibodies used were Pyk2 (Cell Signaling), phospho-Pyk2 (Tyr402) (Cell Signaling), FAK (Cell Signaling), phospho-FAK (Tyr397) (Cell Signaling), and β-actin (Sigma). Signal intensities were quantified by a luminescent image analyzer (Fujifilm).

MM cells were cultured with or without stimuli; the cells were then harvested, washed, lysed, and stained by using primary antibodies to the following proteins: PARP (#9542), FAK (#3285), Pyk2 (#3292), Phospho-Pyk2 (Tyr402) (#3291), GSK-3-α/β (#5676), p-GSK-3-α/β (Ser²¹/Ser⁹) (#9331), cyclin D1 (#2978) (all Cell Signaling Technology); c-myc (OP10L; EMD Biosciences, San Diego, CA); Actin-HRP (sc-1615; (Santa Cruz Biotechnology); β-catenin (CAT5-H10; Invitrogen Corporation, Carlsbad, CA). Standard chemiluminescence was used to evaluate protein expression.