Anti matrix metalloproteinase 2 mmp 2

CoCl₂, AMD3100 and NAC were purchased from Sigma (St. Louis, MO). The pAkt inhibitor LY294002 and the pERK1/2 inhibitor PD98025 were purchased from Cell Signaling Technology (Beverly, MA). The mTOR inhibitor rapamycin and proliferation inhibitor Epothilone B were obtained from Selleck Chemicals (Houston, TX). The anti-HIF-1α and anti-CXCR4 rabbit monoclonal antibodies were purchased from Abcam (Cambridge, MA). The anti-phospho-Akt, anti-Akt, anti-phospho-ERK1/2, anti-ERK1/2, anti-matrix metalloproteinase 2 (MMP-2), anti-MMP-9 and anti-GAPDH monoclonal antibodies were obtained from Cell Signaling Technology (Beverly, MA). Human CXCR4 short hairpin RNA and HIF-1α small-interfering RNA were obtained from Genepharma (Shanghai, China). The transfection reagent was purchased from Tiangen (Beijing, China).

Primary antibodies used in this work were rabbit polyclonal anti-HA (ab9110) from Abcam (Cambridge, UK); mouse monoclonal anti-RAB7A (sc376362), anti-vimentin (sc6260), anti-p-αPAK (sc-135755), anti-αPAK (sc-166887), anti-cofilin-1 (sc-53934), anti-caspase 9 (sc-56073), rabbit polyclonal anti-myc (sc-789), anti-p-AKT 1/2/3 (sc7985-R), anti-AKT 1/2/3 (sc8312), anti-p-PKA α/β/γ cat (sc32968), anti-PKA (sc903), and ROCK-2 (sc5561), from Santa Cruz Biotechnology (Santa Cruz, CA, USA); mouse monoclonal anti-β-catenin (610154) from BD Transduction Laboratories (San Jose, CA, USA); rabbit polyclonal anti-cleaved caspase 9 (#7237), anti-NF-kB p65 (#8242), anti-matrix metalloproteinase 2 (MMP2) (#4022), anti-p-vimentin S38 (#13614), and anti-p-p65/RelA (Ser536) (#3033) from Cell Signaling Technology (Leiden, The Netherlands); mouse monoclonal anti-tubulin (T5168) from Sigma-Aldrich and rabbit polyclonal p-ROCK2 (GTX122651) from GeneTex (Irvine, CA, USA). Secondary antibodies conjugated to fluorochromes (for immunofluorescence analyses) or horseradish peroxidase (HRP, for immunoblot analyses) were from Invitrogen (Carlsbad, CA, USA) or Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The total proteins from mice tumor tissues and SCC9 cells were isolated, and the protein concentrations were determined through a bicinchoninic acid assay. The proteins from whole-cell lysates were used for western blot using standard techniques. Anti-RASSF-1A (1:1000), anti-caspase3 (1:500), anti-CyclinD1 (1:500), anti-CyclinD2 (1:500), anti-Matrix Metalloproteinase-2 (MMP2) (1:1000), anti-GAPDH (1:1000) and anti-beta-actin (1:1000) antibodies (Cell Signaling Technology, Danvers, MA, USA) were used. The co-IP assay was performed using Protein A/G agarose (Beyotime, China), following the manufacturer’s instructions and normal rabbit anti-IgG (Abcam, UK) was used as a control antibody. Harvested samples containing input (protein pretreated with nothing), IgG (protein pre-treated with A/G agarose and rabbit anti-IgG), anti-RASSF-1A (protein pre-treated with A/G agarose and rabbit anti-RASSF-1A), and anti-CyclinD1 (protein pre-treated with A/G agarose and rabbit anti-CyclinD1) were analyzed by western blot.