Verification of the TβRII^-/- knock out in thymocytes from recipients of TβRII^-/- bone marrow at 3 weeks after transplantation. (A) Reconstitution of thymocytes as determined by flow cytometry and staining with anti-Ly5.2 antibodies against cells of donor origin. The diagram shows mean values of reconstitution (% of Ly5.2⁺ thymocytes, n = 8). C = TβRII^+/- controls (n = 8). (B) SQ-PCR analysis of TβRII^-/- CD4^-CD8^- double-negative (DN^-/-) thymocytes from bone marrow recipients at 3 weeks after transplantation, to determine Cre/lox recombination efficiency. The top panel shows the absence of the flox allele among DN^-/- thymocytes, whereas the bottom panel shows the presence of the null (-) allele. PCR products were generated from 3 dilutions of each DNA template. The PCR reactions are specific for the flox and wild-type (+) alleles (top panel) and the null allele (bottom panel). Both PCRs were run on the same DN^-/- DNA preparation. Band intensities among the dilutions of control DNA (+/flox and +/-) indicate that the amount of template chosen is proportional to the amount of PCR product. The wild-type band seen in the DN^-/- 2-ng lane of the flox/wild-type PCR is derived from a minor fraction of contaminating wild-type (wt) cells surviving irradiation (< 3% as seen in A). The 2-fold amount of control DNA in the PCR to detect the null allele was used to compensate for heterozygosity. L = 100-bp ladder. (C) Test of antiproliferative response of TβRII^-/- or control (C = TβRII^flox/flox) thymocytes to TGFβ1, in the presence of ³H-thymidine. Proliferation is measured by incorporation of ³H-thymidine, which is monitored as radioactive decay (cpm). (D) Western blot analysis to detect the presence of phospho-Smad2 (indicated by the arrow) in thymocytes following stimulation with TGFβ1, indicated by +.C = control thymocytes (TβRII^flox/flox). Trace amounts of phospho-Smad2 in TβRII^-/- thymocytes are most likely due to the contamination with approximately 5% wild-type cells.