pyzd-4409  (MedChemExpress)

Journal: Communications Biology

Article Title: Proteolysis targeting chimera (PROTAC)-driven antibody internalization of oncogenic cell surface receptors

doi: 10.1038/s42003-024-07439-0

Figure Lengend Snippet: Antibody internalization was evaluated in the presence of endocytosis inhibitors (Dyngo-4a and Pitstop2) and ubiquitin-proteasome system inhibitors (TAK-243, PYZD-4409, and MG132). a BT-474 and SKBR3 cells were assayed by in vitro live-cell imaging for Tmab internalization and PROTAC SJF1528 in presence of the dynamin mediated endocytosis inhibitor Dyngo-4a at (30 and 15 µM). b SKBR3 and HCC827 GR6 cells were also assayed for Tmab or anti-MET internalization and PROTAC SJF1528 or 48-284, respectively, in presence of the Uba1 inhibitors TAK-243 and PYZD-4409. c Time course evaluation of the internalized Tmab by Western blots with antibodies specific against light and heavy human IgG chains and HER2 expression on SKBR3 cells treated with or without SJF1528 200 nM and Tmab 2 µg/mL, and SJF1528 200 nM with Tmab 2 µg/mL and MG132 5 µM. d Representative images of immunofluorescence staining of BT-474 cells assayed overnight with SJF1528 200 nM and labeled Tmab 2 µg/mL or SJF1528 200 nM with Tmab 2 µg/mL, MG132 5 µM and a secondary antibody for human IgG–FITC conjugated. Data shown here are representative experiments, every condition has been done in triplicate and lines and error bars represent the medians and SEM. Individual data points are available in the file and the uncropped Western blots are in the Supplementary figures. Statistical significance was evaluated with GraphPad Prism 10 by unpaired t -test and two-tailed p value. **** P < 0.0001.

Article Snippet: Both, the dynamin I/II or clathrin inhibitors Dyngo-4a and Pitstop2 (Med Chem Express HY-13863 and HY-115604, respectively) and Uba1 inhibitors TAK-243 and PYZD-4409 (Med Chem Express HY-100487 and HY-13297, respectively) were added to the cells at the same time that labeled antibodies Tmab and anti-MET were added, and internalization evaluated by acquiring images with IncuCyte every 30 min for 24–48 h as before.

Techniques: In Vitro, Live Cell Imaging, Western Blot, Expressing, Immunofluorescence, Staining, Labeling, Two Tailed Test

Journal: Infection and Immunity

Article Title: GABARAPL2 Is Critical for Growth Restriction of Toxoplasma gondii in HeLa Cells Treated with Gamma Interferon

doi: 10.1128/IAI.00054-20

Figure Lengend Snippet: GABARAPL2 accumulated on T. gondii PVs by an IFN-γ and ubiquitin-dependent mechanism. (A to D) HeLa cells were left unactivated or activated with IFN-γ for 24 h and treated with the E1 inhibitor PYR-41 or PYZD-4409 for 6 h. Cells were infected with parasites (MOI = 10) and harvested at 4 h postinfection. Cells were stained with antibodies against GRA7 and ubiquitin or GFP, followed by secondary antibodies conjugated to either Alexa Fluor 594 (red) or 488 (green). The average number of vacuoles was determined from at least 100 vacuoles on three individual coverslips. Values are the mean ± SD of two independent experiments, and statistical analysis was done by t tests. ** P < 0.01. (E and F) GABARAPL2KO cells were left unactivated or were activated with IFN-γ for 24 h, infected with the parasite (MOI = 10), and harvested at 4 h postinfection. Cells were treated as above. The average number of vacuoles was determined from at least 100 vacuoles on three individual coverslips. Values are the mean ± SD of three independent experiments, and statistical analysis was done by t tests; ns, not significant.

Article Snippet: The ubiquitin E1 inhibitors PYR-41 (11117) and PYZD-4409 (15183) were purchased from MedChem Express.

Techniques: Infection, Staining