Hrp conjugated donkey anti goat antibodies

Anti-NOSTRIN (ab116374) antibody was purchased from Abcam (Cambridge, MA, USA) and was used in 1:100 dilutions. Other primary antibodies were purchased from either Cell Signaling Technology (CST, Beverly, MA, USA) and used in 1:1000 dilutions or from Santa Cruz Biotechnology (SCBT, Dallas, TX, USA) and used in 1:250 dilutions. Antibodies obtained from CST are as follows: anti-HIF1α (14179), anti-INTEGRIN α5 (98204), ANTI-INTEGRIN β3 (4702), anti-VEGF Receptor-2/KDR (9698), anti-BECLIN1 (3495), anti-LC3A/B (12741), anti-SQSTM1 (23214) and NF-κB Pathway Sampler Kit (9936). Others, procured from SCBT are anti-TACE/ADAM-17 (sc-6416), anti-COL18A1 (sc-16651), anti-FLT-1 (sc-316), anti-FIBRONECTIN (sc-6952), anti-MMP-2 (sc-10736), anti-PGF (sc-1883), anti-uPA (sc-14019), anti-TIE-2/TEK (sc-324), and anti BCL2 (sc-7382). HRP conjugated goat anti-rabbit and rabbit anti-mouse antibodies were purchased from Cell SignalingTechnology, and HRP conjugated donkey anti-goat antibodies were purchased from Santa Cruz Biotechnology. Both the secondary antibodies were used in 1:2000 dilutions.

Single-cell suspensions from BM of wild-type (WT) and miR-128-2 TG mice were prepared for staining with specific fluorescence-conjugated antibodies. PreproB (B220⁺IgM⁻) and CLP (Lin⁻ckit^intSca1⁺) cells were sorted using FACS ArrayII. Sorted cells were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Equal concentrations of protein were separated on a denaturing sodium dodecyl sulfate–10% polyacrylamide gel and then transferred to nitrocellulose by electroblotting. Proteins were detected with a 1:1000 dilution of rabbit anti-MALT1 (#2494s, Cell Signaling Inc., Danvers, MA, USA) or goat anti-A2B (R-20) (#Sc-7507, Santa Cruz Biotechnology Inc., Dallas, TX, USA) and a 1:5000 dilution of HRP-conjugated anti-rabbit antibodies (#7704, Cell Signaling Technology, MA, USA) or HRP-conjugated donkey anti-goat antibodies (#Sc2020, Santa Cruz Biotechnology Inc., Dallas, TX, USA). GAPDH (#14C10, Cell Signaling Inc., Danvers, MA, USA) was used as loading reference. HRP was detected with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Waltham, MA, USA).