pcag nls ha bxb1 plasmid  (Addgene inc)

Journal: Cell reports

Article Title: HP1γ self-assembles and cooperates with KAP1 in repression of long noncoding RNA AI662270 in ESCs

doi: 10.1016/j.celrep.2025.116874

Figure Lengend Snippet: (A) AI662270 expression was measured in HP1α −/− , HP1β −/− , HP1γ −/− , and KAP1 −/− mouse ESCs using quantitative reverse transcription PCR (qRT-PCR). Data are presented as average between two independent biological replicates. (B) Generation of the entry mouse ESC line. The attP sequence (MIN tag) is inserted directly after the transcription start site of HP1γ. Schematic illustrating the CRISPR-Cas9-mediated genome editing strategy, with the gRNA and PAM sequences highlighted. The donor single-strand DNA contains the MIN tag sequence with a HincII restriction cut site for screening and homology arms flanking the translational start site. The positions of screening PCR primers are indicated, which produce 509 and 557 bp products in WT and HP1γ attP/attP cells, respectively. (C) PCR-based validation of two individual HP1γ attP/attP ESCs using primers indicated in (B), followed by HincII digestion. PCR products showing a reduced size after HincII digestion are considered positive. (D) Schematic outlining the Bxb1-mediated recombination strategy used to generate the HP1γ −/− ESC line. A cassette containing the attB site, RFP, a stop codon, and a polyA signal was inserted directly after the transcription start site by attP-attB recombination to produce HP1γ −/− ESCs. (E and F) Characterization of two individual HP1γ −/− ESC clones, A3 and C3, by quantitative RT-PCR analysis (E). Data are presented as mean ± standard deviation (SD) from three technical replicates. Western blot analysis to detect HP1γ in the two individual HP1γ −/− clones (F). The tubulin blot was used as a loading control. See also 1 and .

Article Snippet: 2 × 10 5 entry HP1γ attP/attP cells were co-transfected with the attB-RFP-Stop-PolyA construct and Bxb1 recombinase (Addgene, #51271).

Techniques: Expressing, Reverse Transcription, Quantitative RT-PCR, Sequencing, CRISPR, Biomarker Discovery, Clone Assay, Standard Deviation, Western Blot, Control

Journal: bioRxiv

Article Title: BAG6 and RNF126 are broadly involved in protein quality control of non-native missense protein variants

doi: 10.64898/2026.02.04.703735

Figure Lengend Snippet: (A) Schematic illustration of the expression system. For single copy expression, a plasmid (vector DNA) encoding GFP-Parkin followed by an internal ribosomal entry site (IRES) and mCherry, was introduced into the landing pad in the HEK293T genome (gDNA) by Bxb1 site-specific recombination. This displaces the cassette of BFP-iCasp9-BlastR separated by 2A stop/start sites (arrowheads). The expressed GFP-tagged Parkin protein variant is subject to PQC degradation (Pacman) resulting in reduced GFP fluorescence, while the mCherry level is constant. Created in BioRender. Hartmann-Petersen, R. (2025) https://BioRender.com/m5by8sy . (B) Comparison of protein levels of wild type Parkin, the R42P variant and empty vector (EV) by SDS-PAGE and western blotting of whole cell lysates. mCherry and β-actin were included as controls. (C) Representative flow cytometry profiles for landing pad cells expressing Parkin wild-type (n = 9.8×10 3 ) and R42P (n = 1×10 4 ) treated with 10 µM bortezomib (BZ) for 16 hours. Untreated wild-type (n = 9.7×10 3 ) and R42P (n = 1×10 4 ) cells are included for comparison. (D) Representative flow cytometry profiles for landing pad cells expressing Parkin wild-type (n = 9.5×10 3 ) and R42P (n = 1×10 4 ) treated with 1 µM TAK243 for 6 hours. The untreated cells (from panel C) are included for comparison.

Article Snippet: The recombinase Bxb1 was expressed from pCAG-NLS-HABxb1 (Addgene #51271) ( ).

Techniques: Expressing, Plasmid Preparation, Variant Assay, Fluorescence, Comparison, SDS Page, Western Blot, Flow Cytometry