Journal: PLoS pathogens

Article Title: Cytolethal distending toxins require components of the ER-associated degradation pathway for host cell entry.

doi: 10.1371/journal.ppat.1004295

Figure Lengend Snippet: Figure 2. Derl2 is required for CDT intoxication. Viability of parental A745TKR cells, retrovirally induced mutant CHO-CDTRC1 cells, and CHO- CDTRC1 cells expressing Derl2 after intoxication with Aa-CDT (a), Hd-CDT (b), Ec-CDT (c) and Cj-CDT (d). Intoxication was performed similar to Fig. 1. (e) Top: representation of the Derl2 open reading frame with boxes representing exons, gray arrows representing primers, and upside down triangles representing proviral insertions. Bottom: agarose gel of genomic PCR from parental A745TKR, CHO-CDTRC1 and CHO-CDTRF1 cells using primers detailed in the diagram. (f) Overexpression of Derl1 does not complement resistance to CDT. Derl2 deficient CHO-CDTRC1 cells expressing empty vector, Derl1, and Derl2 were intoxicated with Hd-CDT, similar to Fig. 1. (g) Derl2 was immunoprecipitated from normalized cell lysates and precipitated proteins analyzed by western blot with anti-Derl2 antibody. (h) CRISPR mediated deletion of Derl2 in HeLa cells causes resistance to Hd- CDT. HeLa cells were transfected with Cas9 DNA and gDNA, followed by selection with G418 and Hd-CDT. Following selection, wildtype and

Article Snippet: Protein-A sepharose beads (Santa Cruz Biotechnology) were washed, blocked with 5% bovine serum albumin (EMD Millipore) and incubated with the lysates for 1 hour at room temperature with agitation.

Techniques: Mutagenesis, Expressing, Agarose Gel Electrophoresis, Over Expression, Plasmid Preparation, Immunoprecipitation, Western Blot, CRISPR, Transfection, Selection

Journal: PLoS pathogens

Article Title: Cytolethal distending toxins require components of the ER-associated degradation pathway for host cell entry.

doi: 10.1371/journal.ppat.1004295

Figure Lengend Snippet: Figure 5. Identification of Derl2 domains required for CDT intoxication. (a–c) CHO-CDTRC1 cells expressing empty vector (squares), Derl1-S (triangles), or Derl2-S (diamonds) were intoxicated in each panel, similar to Fig. 1, and compared to derlin variants indicated below. Anti-DERL1 (a) or anti-Derl2 (b, c) western blot of S-protein agarose precipitated protein from normalized cell lysates show expression levels of chimeric derlins. Cartoons depict Derl1 (black) and Derl2 (grey) sequences in each chimera. (a) CHO-CDTRC1 cells expressing Derl1-S (triangles, #1) or Derl21–187:Derl1189–251-S tag (circles, #2) were challenged with Hd-CDT. (b) CHO-CDTRC1 cells expressing Derl2-S (diamonds, #1), Derl21–112: Derl1114–121:Derl2120–239-S (circles, #2) or Derl21–161:Derl1163–171: Derl2171–239-S (inverted triangles, #3) were intoxicated as above. (c) CHO-CDTRC1 cells expressing Derl2-S (diamonds, #1), Derl11–88:Derl288–239-S (open boxes, 2), Derl11–138:Derl2138–239-S (open triangles, #3) or Derl11–162:Derl2162–

Article Snippet: Protein-A sepharose beads (Santa Cruz Biotechnology) were washed, blocked with 5% bovine serum albumin (EMD Millipore) and incubated with the lysates for 1 hour at room temperature with agitation.

Techniques: Expressing, Plasmid Preparation, Western Blot