er markers include mcherry mch sec61β  (Addgene inc)

Journal: Communications Biology

Article Title: Identification of SARS-CoV-2 inhibitors targeting Mpro and PLpro using in-cell-protease assay

doi: 10.1038/s42003-022-03090-9

Figure Lengend Snippet: a , b Constructs designed for the ICP assay containing mEmerald with nuclear localization signal (NLS), cleavage site for Mpro or PLpro, Zika virus NS2B followed by Mpro ( a ) or PLpro (amino acids 1541–1855 of nsp3) ( b ). c – f Localization of mEmerald-NLS (green) and ER marker mCherry-Sec61 β (red) at 6 h post-transfection and stained with nuclear stain Hoechst 33342 dye (blue), in ICP assay. c Cells transfected with ICP construct A, d cells transfected with ICP construct A with inactive Mpro mutant (C145A). e Cells transfected with ICP construct B, f cells transfected with ICP construct B with inactive PLpro mutant (C1651A).

Article Snippet: In-cell protease assay plasmids were co-transfected with the ER marker mCherry Sec61 β C1 (Addgene Plasmid # 90994) to determine the colocalization of uncleaved protein with the ER .

Techniques: Construct, Virus, Marker, Transfection, Staining, Mutagenesis

Journal: PLoS ONE

Article Title: Involvement of Endoplasmic Reticulum Stress in TULP1 Induced Retinal Degeneration

doi: 10.1371/journal.pone.0151806

Figure Lengend Snippet: P1 WT mice retinas were co-transfected with GFP-fused (green) plasmids and a mCherry tagged ER reporter (mCh-Sec61b: red). Retinas were harvested and sectioned at P30 (A) Confocal images of retinas show co-localization (merge: yellow) of GFP-fused mutant D94Y-TULP1 and F491L-TULP1 with mCh-Sec61b, the ER resident protein, in the inner segments. Scale bar = 10μM. (B-C) Activation of the ER-UPR stress markers in transgenic mice expressing mutant TULP1 protein. qRT-PCR indicates a 1.3–5 fold increase of multiple ER-UPR stress markers in mutant D94Y-TULP1 (B) or mutant F491L-TULP1 (C) retinas vs WT-TULP1 injected retinas. qRT-PCR analysis was normalized to GAPDH (as a mRNA quantity control) and GFP (as a transfection efficiency control). The ΔΔCt method was employed to calculate fold changes. Samples were assayed in triplicates for each ER-UPR marker and qRT-PCR experiments were repeated twice. Statistical significance was assessed by using the two-tailed Student’s t -test. * = p<0.001; n.s. not statistically significant.

Article Snippet: For co-localization studies, WT or mutant TULP1 plasmids were co-electroporated with the mCherry-tagged ER-marker Sec61b plasmid DNA (mCherry-Sec61b, a gift from Dr. Gia Voeltz (Addgene #49155)).

Techniques: Transfection, Mutagenesis, Activation Assay, Transgenic Assay, Expressing, Quantitative RT-PCR, Injection, Control, Marker, Two Tailed Test