a,b) Immunoblot analysis of LAMP-2A in total lysates (a) and quantitative PCR analysis of Lamp2a mRNA (b) of naive CD4+ T cells and TH1 and TH2 cells following activation with anti-CD3 and anti-CD28.figf1(a,b) Immunoblot analysis of LAMP-2A in total lysates (a) and quantitative PCR analysis of Lamp2a mRNA (b) of naive CD4+ T cells and TH1 and TH2 cells following activation with anti-CD3 and anti-CD28. β-actin (a) serves as a loading control throughout; mRNA results (b) are presented relative to those of resting cells. (c) Immunofluorescence microscopy of LAMP-2A (green) and nuclei (stained with the DNA-binding dye DAPI; blue) in resting TH1 cells (Rest) and TH1 cells activated for 24 h as in a,b (Act) (left), and quantification of lysosomes in cells as at left (right). Original magnification, ×630; scale bar, 10 μm. *P = 0.013 (t-test). (d) Electron microscopy (left) of resting TH1 cells and TH1 cells activated for 24 h as in a,b (top row); below, enlargement of areas outlined above, showing individual lysosomes (arrows). Original magnification (top row), ×12,000; scale bars (bottom row), 1 μm. Right, morphometric analysis of cells as at left. *P = 0.037 and **P = 0.034 (t-test). (e) Distribution of KFERQ-PA-mCherry-1 in TH1 cells transiently transfected with a CMA reporter vector, photoactivated, and left unstimulated (Rest) or stimulated with anti-CD3 and anti-CD28 in the presence (3-MA) or absence (vehicle (veh)) of 3-methyladenine (left); arrows indicate mCherry+ puncta. Original magnification, ×630. Right, quantification of the results for the cells at left. *P 0.05 (analysis of variance with Tukey's post-test). (f) Immunoblot analysis of hsc70 in lysosomal fractions of resting (Lys-R) and stimulated (Lys-A) TH1 cells and in cytosolic fractions of TH1 cells activated as in a,b (Cyto); LAMP-2A and LAMP-1 serve as controls. (g) Immunoblot analysis of LAMP-2A in resting TH1 cells and TH1 cells activated in the presence (NAc) or absence (Veh) of N-acetylcysteine. (h) Quantitative PCR analysis of Lamp2a mRNA in cells as in g (results presented as in b). *P = 0.007 (t test). (i) Luciferase activity in TH1 cells transiently transfected with a vector containing a luciferase reporter under the control of the Lamp2 proximal promoter and stimulated for 0, 12 or 24 h with plate-bound anti-CD3 and anti-CD28; results are presented relative to those of resting cells (time 0). (j) Luciferase activity in TH1 cells transiently transfected as in i and left unstimulated (control (Ctrl)) or stimulated for 16 h with 50 μM paraquat (Pq) or 1 mM H2O2; results are presented in relative light units (RLU). (k) Immunoblot analysis of LAMP-2A in total lysates of resting TH1 cells (−) and TH1 cells activated for 24 h as in a,b (+), transfected with a control nontargeting siRNA (with a scrambled sequence (Scr)) or siRNA specific for mRNA encoding Duox1 (either of two siRNAs: Dx(1) and Dx(2)) or RISP (Uq(1)). (l) Immunoblot analysis of LAMP-2A in lysates of resting or activated TH1 cells in the presence (right half) or absence (left half) of cyclosporine A (CsA). (m) Chromatin-immunoprecipitation analysis of the recruitment of NFAT1 to the promoter of Lamp2 or Adad1 (negative control) (left) or to Il2 (positive control) (right) in resting and activated TH1 cells; results are presented relative to input chromatin precipitated with anti-NFAT1.