Peroxidase labeled anti mouse secondary antibody

Strains XC.052 and XC.109 were grown to mid-log phase in 500 ml of M9 minimum medium plus 0.3% (wt/vol) glucose, 0.1% (wt/vol) Casamino acids with limited (1.6 nM) or excess (40 μM) biotin. The soluble cell extracts were collected and equal amounts of protein were loaded onto a nickel affinity chromatography column. The proteins were purified as above and were analyzed by SDS-PAGE. Equal volumes of soluble proteins were loaded and separated on a 12% SDS-polyacrylamide gel and transferred by electrophoresis to Immobilon-P membranes (Millipore) for 15 min at 15 V. The membranes were preblocked with TBS buffer (100 mM Tris base and 0.9% NaCl, pH 7.5) containing 0.1% Tween 20 and 5% nonfat milk powder. The membranes were probed for 1 h with an anti-His₆ protein primary antibody (ThermoFisher Scientific) diluted 1:2,000 in the above buffer. Following incubation with a peroxidase labeled anti-mouse secondary antibody (diluted 1:5000; GE Healthcare Life Sciences), the labeled proteins (His₆-tagged BioH) were detected using Quantity One software.

The anti-NG2 antibody (1:100; sc-166251) was from Santa Cruz Biotechnology (Heidelberg, Germany). The anti-α-tubulin antibody (1:1,000; 66031) was from Proteintech Germany GmbH (St. Leon-Rot, Germany). The antibodies anti-Akt1/2/3 (1:100; 4685), anti-pAkt (1:100; 4060) and anti-c-Myc (1:50; 5605) were from Cell Signaling (Frankfurt am Main, Germany). The anti-ERK1/2 antibody (1:100; ab115799), anti-pERK1/2 antibody (1:100; ab50011) and anti-PTEN antibody (1:50; ab31392) were from Abcam (Cambridge, UK). The peroxidase-labeled anti-rabbit antibody (1:1000; NIF 824) and peroxidase-labeled anti-mouse secondary antibody (1:1,000; NIF 825) were from GE Healthcare (Freiburg, Germany). The PE-labeled anti-chondroitin sulfate proteoglycan 4 antibody (1:30; NG2; 562415), PE-labeled anti-ITGB1 antibody (1:30; 556049), PE-labeled anti-PDGFRα antibody (1:30; 556002), and PE-labeled anti-PDGFRβ antibody (1:30; 55821) were from BD Biosciences (Heidelberg, Germany).