Journal: bioRxiv

Article Title: Prediction and validation of host cleavage targets of SARS-CoV-2 3C-like protease

doi: 10.1101/2022.01.17.476677

Figure Lengend Snippet: A) Western blot of in vitro cleavage of recombinant cadherin with purified 3CLPro. 1uM of purified 3CLPro was incubated with 2ug of recombinant CDH6 or CDH20 (C-terminal His-Tag) in a 50uL reaction for 1hr. Shown are cleavage sites within the recombinant fragment, with amino acid positions displayed for the full length proteins. Western blots showing staining against the C-terminus (His-Tag) of each protein and 3CLPro. Recombinant proteins are a mixture of glycosylated (∼90kDa) and unglycosylated (∼65kda), corresponding to cleavage fragments of ∼62kDa and 40kDa (respectively). B) Western blot of in vitro cleavage of purified human alpha thrombin (IIa). Diagram shows amino acid position of unprocessed prothrombin. Position of cleavage site shown with respect to epitope of antibody used for western blot. 1uM of purified 3CLpro was incubated with 2ug alpha thrombin overnight under reducing conditions, with or without the 3CLPro inhibitor GC376 (1uM). C) In vitro cleavage of purified recombinant NOTCH1 fragment (aa2280-2550) with a N-terminal His-Tag. Reactions were done with 1uM of purified 3CLPro for 1hr. Diagram shows position of cleavage within the NOTCH1 fragment, with amino acid positions corresponding to the full length protein. Epitope regions showed for antibody with epitope C-terminal to the cleavage site. Full length size is ∼29kDa, with N and C-terminal fragments of 4kDa and 25kDa (respectively). D) Cellular cleavage of NOTCH1. Western blots show lysates of hIPSC cardiomyocytes expressing 3CLPro or catalytically inactive C145A variant for 48h. Cleavage site position within the intracellular fragment of NOTCH1 shown, as well as epitope for antibody used in western blot. For all statistics shown, * = p ≤ 0.05 , ** p ≤ 0.01, ***p ≤ 0.001, **** = p ≤ 0.00001

Article Snippet: Recombinant proteins were purchased commercially: NOTCH1 (Origene, Cat# TP762041), CDH6 (ACROBiosystem, Cat# CA6-H5229), CDH20 (R&D, Cat# 5604-CA-050).

Techniques: Western Blot, In Vitro, Recombinant, Purification, Incubation, Staining, Expressing, Variant Assay

Journal: Journal of visualized experiments : JoVE

Article Title: Using microarrays to interrogate microenvironmental impact on cellular phenotypes in cancer.

doi: 10.3791/58957

Figure Lengend Snippet: The full list of ECM proteins and conditions that are used in the MEMA experiments. The uniprot ID, stock concentrations, and final working concentrations are provided. In some instances, the printed condition represents a protein complex or a combination of multiple proteins, which is indicated in the notes column.

Article Snippet: Cadherin-20 (CDH20) , R & D Systems , 5604-CA , ECM.

Techniques: Concentration Assay

Journal: Journal of visualized experiments : JoVE

Article Title: Using microarrays to interrogate microenvironmental impact on cellular phenotypes in cancer.

doi: 10.3791/58957

Figure Lengend Snippet: Materials

Article Snippet: Cadherin-20 (CDH20) , R & D Systems , 5604-CA , ECM.

Techniques: Imaging, Inverted Microscopy, Microarray, Electron Microscopy