Sc 9935

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).

Protein was extracted after homogenizing mammary gland tissue, and the concentrate was assayed using the Bradford method [28 ]. Proteins were separated using SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, USA). The membrane that included proteins <50 kDa was incubated with a specific primary antibody, polyclonal antibody raised in goat (sc-23016, Santa Cruz, USA), and the membrane that included proteins >50 kDa was normalized against β-tubulin and incubated with a polyclonal goat antibody (sc-9935, Santa Cruz, USA). Membranes were incubated overnight at 4°C and re-probed with a horseradish peroxidase-conjugated Affinipure rabbit anti-goat secondary antibody (E030130-01, Earthox LLC, San Francisco, CA). Protein bands were visualized using a chemiluminescent agent (BeyoECL plus, Shanghai, China). Membranes were scanned using a chemiluminescence imager, Image Quant LAS 4000 (GE, USA), and band intensities were densitometrically evaluated using Quantity One software (Bio-Rad, USA). Signal intensities of the samples are expressed as a percentage of the reference sample.

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).