Figure 3

Western blot analysis of RON and TIE1 expression and activation in MBL and HL cell lines. Lysates corresponding to approximately 1 × 10⁵ cells were loaded per lane and after blotting to membranes probed with the anti-RON (a) and anti-TIE1 (b) antibodies used for IHC. For RON the HL cell lines L428 and L1236 were included as positive and KMH2 as negative control. Although nearly similar amounts of RON transcripts were observed in Karpas1106P and MedB-1 with real-time RT–PCR (data not shown) only for MedB-1 strong RON protein expression was observed. TIE1 expression was only observed in Karpas1106P and the HL cell line KMH2, in line with real-time PCR analysis, where among the HL cell lines L1236, L428, KMH2 and HDLM2 only KMH2 showed TIE1 expression (data not shown). Loading of equal amounts of proteins was controlled with an anti-actin antibody. For analysis of the activation status of the RTKs, cell lines were incubated for 2 h with 2 mM Na₃VO₄ before lysis to enrich tyrosine-phosphorylated proteins. P-Y-proteins were immunoprecipitated from lysates of 1 × 10⁷ cells with a pan-p-Y antibody (4G10)(lanes B in (c) and (d) and 1/10 of the lysates were analysed with RON (c) and TIE1 (d) specific antibodies. Normal mouse Ig was used as negative control (lanes A in (c) and (d)).