Fig 1: GP63 alters nuclear envelope protein levels and of proteins involved in nuclear transport.(A and B) Analysis of purity of LC-MS/MS samples. (A) LM1 MΦ cells were infected as indicated with L. major species (WT, GP63-/-) or L. mexicana for 2 hrs. Total protein lysate (TPL), whole nuclei (WN), nuclear envelope (NE), and nucleoplasm (NP) samples were generated. An antibody against GP63 was used to monitor its presence. Antibodies against KDEL (ER marker), PGK1 (cytoplasmic marker), and Actin (loading control) were used as controls. (B) Silver staining of infected samples. LM1 MΦ cells were infected as indicated with L. major species (WT, GP63-/-) or L. mexicana for 2 hrs. Whole nuclei (WN) and nucleoplasm (NP) lysates were generated. Parasite lysates were used as controls (Par Lys). (C) Impact of Leishmania infection on the proteins of the nuclear envelope and the nuclear transport machinery. INM: Inner Nuclear Membrane; ONM: Outer Nuclear Membrane. (D) Confirmation of the LC-MS/MS results for WN samples by western blot. LM1 MΦ cells were infected as indicated with L. major species (WT, GP63-/-) or L. mexicana for 2 hrs. Total protein lysates (TPL, left panel) and whole nuclei (WN, middle panel) lysates were generated. Parasite lysates were used as controls (Par Lys, right panel). Specific antibodies were used to monitor Nup93, RanGAP1 and Kpnβ1. Antibody against GP63 was used to monitor its presence. (E) Confirmation of the LC-MS/MS results for NP samples by western blot. LM1 MΦ cells were infected as indicated with L. major species (WT, GP63-/-) or L. mexicana for 2 hrs. Total protein lysates (TPL, left panel) and nucleoplasm (NP, middle panel) lysates were generated. Parasite lysates were used as controls (Par Lys, right panel). Specific antibodies were used to monitor Kpna3 and Kif1B. Antibody against GP63 was used to monitor its presence.
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