PTPRK protein expression inversely correlates with STAT3 activation. (A) Immunohistochemical staining of paraffin sections of representative NKTCL primary tumors with high (NL11) and low (NL22) PTPRK expression using a rabbit polyclonal PTPRK (H75) antibody raised against amino acids 27-101 mapping within an N-terminal extracellular domain of human PTPRK (brown cellular membranous staining between the cell-cell contact and faint cytoplasmic staining is indicated by arrows), and a phospho-STAT3^Tyr705 antibody (brown nuclear staining is indicated by arrows). The immunostaining demonstrated that PTPRK protein expression inversely correlated with phospho-STAT3^Tyr705 levels in NKTCL primary tumors. Original magnification ×400. (B) Western blot analyses of PTPRK and phospho-STAT3^Tyr705 expression in PTPRK nonexpressing NKYS cells with reconstitution of PTPRK expression (i) and in PTPRK-expressing SNK6 cells with partial knockdown of PTPRK expression (ii), using the indicated antibodies. Restoration of PTPRK expression significantly decreased phospho-STAT3^Tyr705 levels in NKYS cells, whereas partial knockdown of PTPRK significantly increased phospho-STAT3^Tyr705 levels in SNK6 cells. (Top of 2Bi) The blot was probed first with a PTPRK antibody and was then stripped and reprobed with the anti-DDK tag M2 antibody to detect the PTPRK-tag fusion protein. β-Actin was used as an internal loading control. The levels of STAT3/phospho-STAT3^Tyr705 proteins were analyzed using STAT3 and phospho-STAT3^Tyr705 antibodies. The entire western blot images showing precursor and mature subunits of PTPRK are presented in supplemental Figures 2A,3. (C) Flow cytometry analyses on (i) PTPRK-transduced (white) vs mock vector-transduced (black) NKYS cells, and (ii) PTPRK-specific shRNA-treated (white) vs control shRNA-treated (red) SNK6 cells using a phospho-STAT3^Tyr705 antibody. (D) (i) PTPRK nonexpressing NKYS cells were infected with a retrovirus expressing PTPRK with a myc-DDK tag at the 3′ end. Double indirect immunofluorescence was performed to detect ectopic PTPRK (detected using an anti-DDK tag M2 antibody, and green fluorescence cellular membranous and cytoplasmic staining) and phospho-STAT3^Tyr705 proteins (red fluorescence nuclear staining). Hoechst 33342 was used to stain the nuclei (blue fluorescence nuclear staining). The images were captured using a Carl Zeiss LSM 510 confocal microscope (original magnification ×600). (ii) PTPRK-expressing SNK6 cells were infected with lentivirus expressing PTPRK-targeting shRNA. Indirect immunofluorescence was performed to detect phospho-STAT3^Tyr705 protein (green fluorescence nuclear staining). Hoechst 33342 was used to stain the nuclei. The image was captured using a Leica Q550CW microscope (original magnification ×400).