resource source identifier antibodies b actin 8h10d10 mouse mab cell signaling technology  (Cell Signaling Technology Inc)

Journal: Trends in biotechnology

Article Title: Without a trace: multiple knockout of CHO host cell hydrolases to prevent polysorbate degradation in biologics.

doi: 10.1016/j.tibtech.2025.04.016

Figure Lengend Snippet: Figure 3. Knockout of Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak1) to rescue reduced harvest viability during fed- batch cultivation. (A) Overview of genes knocked out in final Bax/Bak1 (BB)-multi-knockout (KO) cell line. Western blots against (B) Bak1 and (C) Bax with cell lysate of parental host cell line (P) and BB-multi-KO clones 1–4. β-actin was used as loading control (also see Figure S4A in the supplemental information online for respective qPCR results after BB KO). Viable cell density (VCD) (D) and viability (E) of comparative ambr15 fed-batch cultivations of parental host cell line with multi-KO clones with and without BB KO. Clones were cultivated in biological duplicates (n = 2); data are mean ± standard deviation. Also see Figure S5B in the supplemental information online for additional data on glucose and lactate metabolism. (F) Apoptosis assay performed on Day 11 of the fed-batch cultivation. Fed-batch data for L KO, LP KO, LPPL KO, LPPLC KO, and LPPLCC KO are shown in Figure S4B,C in the supplemental information online. Increased luminescence indicates increased apoptotic signaling. n = 2; data are mean ± standard deviation. (A) Created with BioRender (biorender.com). Abbreviations: Ces, carboxylesterase; InDels, nucleotide insertions and deletions; Iah1, isoamyl acetate hydrolyzing esterase 1; Lipa, lipase A; Lpl, lipoprotein lipase; Pla2g7, platelet-activating factor acetylhydrolase; Pla2g15, phospholipase A2 group XV; Ppt1, palmitoyl-protein thioesterase 1.

Article Snippet: Reagent or resource Source Identifier Antibodies β-Actin (8H10D10) Mouse mAb Cell Signaling Technology Cat#3700 Anti-Bak antibody Rabbit mAb Sigma-Aldrich Cat#B5897 Anti-Bax antibody [E63] Rabbit mAb Abcam Cat#AB32503 Donkey anti-Rabbit IRDye 800CW Licor Cat#925-32211 Goat Anti-Mouse IRDye 680RD Licor Cat#926-68070 Chemicals, peptides, and recombinant proteins Zinc finger nuclease pair targeting exon 7 of Lipoprotein lipase (LPL) Sigma-Aldrich N/A QuickExtract DNA Extraction Solution Lucigen Cat#101098 (Supplier Biozym) Alt-RTM Sp.

Techniques: Knock-Out, Western Blot, Clone Assay, Control, Standard Deviation, Apoptosis Assay