Biochemical characterization and cellular localization of a formalin‐resistant melanoma‐associated antigen reacting with monoclonal antibody NKI/C‐3

Abstract

A monoclonal antibody (MAb NKI/C-3) produced against a purified membrane preparation of human melanoma cells reacts preferentially with sections of formaldehyde-fixed and paraffin-embedded tissues of melanoma, nevocellular nevi, carcinoids and medullary carcinomas of the thyroid. NKI/C-3 did not react with basal-cell carcinoma, brain tissue or brain tumors, and in only 14/196 other tumors was a clear cross-reactivity observed, e.g. with prostate carcinomas and a minority of primary breast, ovarian, lung and clear-cell carcinomas. This antibody was used in an immuno-electron microscopic study for the cellular localization of the antigen. The antigen was dispersed in the cytoplasm of melanoma cells, and more concentrated inside vacuoles and sometimes also on the melanosomes. Occasionally, the antigen was seen on the cell surface. The nature of the antigen was determined in an enzyme immunoassay (EIA). It was found that the antigen is a glycoprotein with a disulfide-dependent configuration that is essential for recognition by the MAb. The antigen was distributed heterogeneously during gel filtration as well as during SDS-polyacrylamide gel electrophoresis in the region of 25-110 kd proteins. A purified antigen preparation that was obtained after affinity chromatography on a column of MAb NKI/C-3 linked to Sepharose 4B contained a carbohydrate:protein ratio of 1:3.5.

Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity

“…Cross-reactivity has been excluded by Western blot analyses with various mRFP-or GFPlabeled fusion proteins. Other antibodies used were: mouse anti-CD63 (28), mouse anti-EEA1 (ab2900; Abcam), mouse anti-V5 and anti-V5-HRP (R96025, R96125; Invitrogen), antiMyc (2278P; Cell Signaling), anti-Myc-HRP (NB600-302H; Novus), anti-HA (12013819001, Roche Applied Science), anti-HA-HRP (ab1190; Abcam), anti-FLAG (M2) and anti-FLAG-HRP (F3165, A8592; Sigma), anti-VPS33A (C1C3) (GTX119416; GeneTex), anti-VPS33b (12195-1-AP, Proteintech), anti-VPS11 (19140-1-AP; Proteintech), anti-TGFBRAP1 (SC-13134 Santa Cruz), anti-VPS41 (13869-1-AP, Proteintech), anti-VPS8 (HPA036871, Sigma), anti-VPS16 (17776-1-AP; Proteintech), and anti-VIPAS39 was a gift of V. Faundez (Center for Translational Social Neuroscience, Emory University, Atlanta). Fluorescent and HRP-conjugated secondary antibodies were obtained from Invitrogen.…”

Purification and Characterization of a 36 kDa Antigen of Mycobacterium leprae

“…Pronase and trypsin digestion, heat treatment and treatment with 8-mercaptoethanol and periodate of the purified antigen were done as described by Vennegoor et al (1985). Treatment with trifluoromethanesulphonic acid was done according to Edge et al (1981).…”

Late endosomal transport and tethering are coupled processes controlled by RILP and the cholesterol sensor ORP1L

“…Crossreactivity has been excluded by Western blot analyses with various mRFP-or GFP-labelled fusion proteins. Other antibodies used were: mouse anti-CD63 (Vennegoor et al, 1985), mouse anti-c-tubulin (Sigma) mouse anti-V5 and anti-V5-HRP (Invitrogen), anti-Myc and anti-Myc-HRP (Novus), anti-HA (Roche) and anti-HA-HRP (Abcam), anti-FLAG (M2) and anti-FLAG-HRP (Sigma) anti-VPS33A (C1C3) (GeneTex), anti-VPS33b (Proteintech), anti-VPS11 (Proteintech), anti-VPS41 (Proteintech), anti-VPS16 (Proteintech), anti-dynein intermediate chain (Sigma, clone 70.1), anti-GAPDH (Sigma), Anti-SPE-39 was a gift of V. Faundez (Center for Translational Social Neuroscience, Emory University, Atlanta). Fluorescent and HRP-conjugated secondary antibodies were obtained from Invitrogen.…”