Skip to main content
. Author manuscript; available in PMC: 2013 Jul 16.
Published in final edited form as: Cancer Res. 2007 Dec 1;67(23):11447–11454. doi: 10.1158/0008-5472.CAN-07-1441

Figure 4.

Figure 4

p56lck in nonlytic TIL is rapidly dephosphorylated upon conjugation with cognate tumor cells. A, phosphorylation status of CD3ζ. Nonlytic or lytic TILs (106) were mixed with 106 MCA38 cells, pulse-centrifuged, and incubated at 37°C for the indicated times. Cell pellets were lysed and immunoprecipitated with 0.002 mg of anti-CD3ζ per sample before immunoblotting with either anti-CD3ζ or antiphosphotyrosine. B, analysis of p56lck pY505 and pY394 levels. Nonlytic or lytic TILs (106) were mixed with 106 MCA38 cells, pulse-centrifuged, and incubated at 37°C for the indicated times before detergent extraction and analysis by immunoblotting as described (9). TIL/tumor conjugate extracts were immunoprecipitated with 0.002 mg of anti-p56lck per sample (clone 3A5, Santa Cruz Biotechnology) and immunoblotted sequentially with 0.0002 mg/mL of p56lck pY394, 0.000175 mg/mL of p56lck pY505 (Cell Signaling), or 0.0002 mg/mL of total p56lck (clone 2102, Santa Cruz Biotechnology). The films were scanned using Adobe Photoshop and densitometry was performed using Kodak 1D3.5.4USB software. For analysis of p56lck, in each case, the signal for the “TIL only” samples was set to “1.0” and the signals for different times of conjugation were compared and shown as that ratio under each gel lane. This experiment was repeated (using the same and several different times of conjugation) five times with equivalent results.