American Society of Hematology

Fig. 4.

$Fig. 4. Association of CD28i with CD28. / (A) Coimmunoprecipitation of CD28i with CD28 in 1% digitonin cell extract. Extracts from CD28i-HA–transfected Jurkat T cells were prepared using 1% Nonidet P40 or 1% digitonin buffer, immunoprecipitated with anti-CD28 Ab (L293) that only reacts with CD28, or an Ab specific for CD43. Immunoprecipitates were fractionated by SDS-PAGE. Western blot membranes were probed with anti-HA Ab. The anti-CD28 only precipitated HA-tagged CD28i from 1% digitonin extract but not from 1% Nonidet P40 extract. In parallel, extracts of CD28i-HA–transfected Jurkat T cells were prepared with 1% digitonin and immunoprecipitated with anti-HA Ab or control Ab. Subsequently, the CD28 C-terminal–specific Western blotting was performed and is shown in the 2 lanes at the right end. Lanes 1 and 6: immunoprecipitated by the control Ab. Lanes 2, 3, and 4: immunoprecipitated by anti-CD28 Ab. Lane 5: immunoprecipitated by anti-CD43 Ab. Lane 7: immunoprecipitated by anti-HA Ab. D indicates digitonin buffer cell extract and N indicates Nonidet P40 buffer cell extract. Filled arrows indicate CD28i-HA in lanes 2, 4, and 7. In lane 7, the open arrow indicates CD28. H indicates the position of the Ig heavy chain, and L indicates the position of the Ig light chain. (B) Detection of HA-tagged CD28i with anti-CD28 C-terminal Ab. One percent digitonin cell extracts of CD28i-HA–transfected Jurkat T cells were immunoprecipitated with anti-CD28 Ab (L293) or anti-HA Ab. Western blot membrane was probed first with anti-HA Ab (left panel). To avoid the background by Ig light chain at approximately 20 kd, the Ig light chain blots were excised before the membrane was reprobed with anti-CD28 C-terminal Ab (right panel). Lane 1: immunoprecipitated with the control Ab. Lane 2: immunoprecipitated with anti-CD28 Ab. Lane 3: immunoprecipitated with anti-HA Ab. In the left panel, the filled arrow indicates CD28i-HA; in the right panel, the filled arrow indicates anti-CD28 C-terminal Ab reactive proteins. L indicates the position of the Ig light chain. (C) Coimmunoprecipitation of CD28i with CD28 in PBL-T 1% digitonin cell extract. One percent digitonin cell extract of PBL-T cells was immunoprecipitated with anti-CD28 Ab (L293). Western blot membrane was probed with anti-CD28 C-terminal Ab. To avoid the background by Ig light chain, the Ig light chain blots were excised prior to film exposure. Lane 1: immunoprecipitated with the control Ab. Lane 2: immunoprecipitated with anti-CD28 Ab. In lane 2, the open arrow indicates CD28. H indicates the position of the Ig heavy chain, and L indicates the position of the Ig light chain. (D) Confocal microscopic study of GFP-fusion proteins of CD28i in Jurkat T cells stained with anti-CD28 Ab. Jurkat T cells were transfected with pEGFP(i-iii) or pEGFP-CD28i (CD28i-GFP) (iv-xii). Sixteen hours later, cells were stained with RPE-conjugated anti-CD28 Ab, which detects CD28 but not CD28i, RPE–anti-CD4, or anti-CD43 Ab-plus Alexa-anti–mouse IgG, then analyzed by confocal microscope. CD28 (i and iv), CD4 (vii) and CD43 (x) were shown in red fluorescence. Only green fluorescence (GFP alone or CD28i-GFP) was detected in the middle panels (ii, v, viii, xi). Red fluorescence and green fluorescence were merged and are shown in the right panels (iii, vi, ix, xii). Only (vi) shows yellow patches resulting from a close association of CD28i-GFP and RPE–anti-CD28 Ab. Transfected plasmids are indicated on the left.$

Association of CD28i with CD28.

(A) Coimmunoprecipitation of CD28i with CD28 in 1% digitonin cell extract. Extracts from CD28i-HA–transfected Jurkat T cells were prepared using 1% Nonidet P40 or 1% digitonin buffer, immunoprecipitated with anti-CD28 Ab (L293) that only reacts with CD28, or an Ab specific for CD43. Immunoprecipitates were fractionated by SDS-PAGE. Western blot membranes were probed with anti-HA Ab. The anti-CD28 only precipitated HA-tagged CD28i from 1% digitonin extract but not from 1% Nonidet P40 extract. In parallel, extracts of CD28i-HA–transfected Jurkat T cells were prepared with 1% digitonin and immunoprecipitated with anti-HA Ab or control Ab. Subsequently, the CD28 C-terminal–specific Western blotting was performed and is shown in the 2 lanes at the right end. Lanes 1 and 6: immunoprecipitated by the control Ab. Lanes 2, 3, and 4: immunoprecipitated by anti-CD28 Ab. Lane 5: immunoprecipitated by anti-CD43 Ab. Lane 7: immunoprecipitated by anti-HA Ab. D indicates digitonin buffer cell extract and N indicates Nonidet P40 buffer cell extract. Filled arrows indicate CD28i-HA in lanes 2, 4, and 7. In lane 7, the open arrow indicates CD28. H indicates the position of the Ig heavy chain, and L indicates the position of the Ig light chain. (B) Detection of HA-tagged CD28i with anti-CD28 C-terminal Ab. One percent digitonin cell extracts of CD28i-HA–transfected Jurkat T cells were immunoprecipitated with anti-CD28 Ab (L293) or anti-HA Ab. Western blot membrane was probed first with anti-HA Ab (left panel). To avoid the background by Ig light chain at approximately 20 kd, the Ig light chain blots were excised before the membrane was reprobed with anti-CD28 C-terminal Ab (right panel). Lane 1: immunoprecipitated with the control Ab. Lane 2: immunoprecipitated with anti-CD28 Ab. Lane 3: immunoprecipitated with anti-HA Ab. In the left panel, the filled arrow indicates CD28i-HA; in the right panel, the filled arrow indicates anti-CD28 C-terminal Ab reactive proteins. L indicates the position of the Ig light chain. (C) Coimmunoprecipitation of CD28i with CD28 in PBL-T 1% digitonin cell extract. One percent digitonin cell extract of PBL-T cells was immunoprecipitated with anti-CD28 Ab (L293). Western blot membrane was probed with anti-CD28 C-terminal Ab. To avoid the background by Ig light chain, the Ig light chain blots were excised prior to film exposure. Lane 1: immunoprecipitated with the control Ab. Lane 2: immunoprecipitated with anti-CD28 Ab. In lane 2, the open arrow indicates CD28. H indicates the position of the Ig heavy chain, and L indicates the position of the Ig light chain. (D) Confocal microscopic study of GFP-fusion proteins of CD28i in Jurkat T cells stained with anti-CD28 Ab. Jurkat T cells were transfected with pEGFP(i-iii) or pEGFP-CD28i (CD28i-GFP) (iv-xii). Sixteen hours later, cells were stained with RPE-conjugated anti-CD28 Ab, which detects CD28 but not CD28i, RPE–anti-CD4, or anti-CD43 Ab-plus Alexa-anti–mouse IgG, then analyzed by confocal microscope. CD28 (i and iv), CD4 (vii) and CD43 (x) were shown in red fluorescence. Only green fluorescence (GFP alone or CD28i-GFP) was detected in the middle panels (ii, v, viii, xi). Red fluorescence and green fluorescence were merged and are shown in the right panels (iii, vi, ix, xii). Only (vi) shows yellow patches resulting from a close association of CD28i-GFP and RPE–anti-CD28 Ab. Transfected plasmids are indicated on the left.

This Feature Is Available To Subscribers Only