Sc 365370

Atg5, Wnt5a and MMP-3 protein levels in cell lysates were determined by western blot analysis. Cells were cultured for 24 h with or without IL-1β (Prepro Tech, Rocky Hill, NJ, USA), lysed and then protein samples were separated on 12% sodium dodecyl sulfate polyacrylamide gels. Western blot analysis was performed using anti-Atg5, anti-LC3, anti-Atg12, anti-Wnt5a, anti-MMP-3, and anti-β-tubulin polyclonal antibodies (sc-8667, sc-398822, sc-68884, sc-365370, sc-6839, and sc-9935, respectively; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The anti-Atg5 and anti-MMP-3 antibodies showed no significant cross-reactivity with other Atgs or MMPs, respectively (data not shown). Visualization and quantification of blotted protein bands were performed with Multi Gauge-Ver3.X software (Fujifilm).

Wnt16, Wnt5a, Wnt5b, Lrp5, Fzd2, Fzd9, Lrp6, Ror1, Ror2, Ryk and MMP-13 protein levels in cell lysates were determined by Western blot analyses. Cells were cultured for 6 h with or without IL-1β, lysed, and then protein lysates were separated on sodium dodecyl sulfate polyacrylamide gels (12%). Western blot analyses were then performed using anti-Wnt16, -Wnt5a, -Wnt5b, -Lrp5, -Fzd2, -Fzd9, -Lrp6, -Ror1, -Ror2, -Ryk, -MMP-13, and -β-tubulin polyclonal antibodies (sc-271897, sc-365370, sc-109464, sc-21390, sc-68328, sc-33509, sc-12363, sc-25317, sc-130867, sc-374174, sc-83080 and sc-9935, respectively; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The anti-MMP-13 antibody showed no significant cross-reactivity with other MMPs (data not shown). Visualization and quantification of blotted protein bands were performed with Multi Gauge-Ver3.X software (Fujifilm, Tokyo, Japan).