rabbit polyclonal anti β actin primary antibody  (Cell Signaling Technology Inc)

Journal: Journal of Lipid Research

Article Title: A novel mutation, Ile344Asn, in microsomal triglyceride transfer protein abolishes binding to protein disulfide isomerase

doi: 10.1016/j.jlr.2024.100725

Figure Lengend Snippet: Ile344Asn does not support lipid transfer and apoB100 secretion. A–C: Mko-3 cells were transfected with 3 μg of plasmid expressing either WT or Ile344Asn MTP. After 48 h, media was replaced with fresh DMEM with 10% FBS. After overnight incubation, cell homogenates (25 μg protein) were used to measure MTP and β-actin (control) protein levels (A) by Western blot analysis and to measure triglyceride (TG) transfer activity (B). Overnight-conditioned media was used to measure apoB-100 secreted by the cells by performing ELISA (C). The amounts of apoB were normalized to cell protein levels. D–F: For lipid transfer assay and MTP:PDI interaction studies, 400 μg protein from the cellular homogenate was incubated overnight at 4°C with anti-Flag M2 affinity gel and the purified MTP protein was eluted with 100 μM Flag peptide in buffer K. Purified protein (20 μl) was separated on SDS-PAGE and probed with anti-hMTP antibodies ( top ), stripped and reprobed with anti-PDI antibody ( bottom ) (D). Amounts of MTP protein bands were quantified using ImageJ and values were used to normalize lipid transfer activities. A different aliquot was used to measure TG and PL transfer activities (E–F). The bars and error bars represent mean ± SD. To calculate the significance one-way ANOVA nonparametric (multiple comparison) or two-way ANOVA was used, ∗∗∗ and ∗∗∗∗ represent P < 0.001 and P < 0.0001, respectively. The data are representative of three independent experiments performed with biological triplicates.

Article Snippet: Rabbit polyclonal anti-β-actin primary antibody (Cell Signaling #4967S) was used to detect the internal reference β-actin.

Techniques: Transfection, Plasmid Preparation, Expressing, Incubation, Control, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Purification, SDS Page, Comparison

Journal: Journal of Lipid Research

Article Title: A novel mutation, Ile344Asn, in microsomal triglyceride transfer protein abolishes binding to protein disulfide isomerase

doi: 10.1016/j.jlr.2024.100725

Figure Lengend Snippet: Increased expression of Ile344Asn did not improve the lipid transfer activity and apoB secretion in Mko-3 cell. Mko-3 cells were transfected with either 2 μg of plasmid expressing WT MTP or 4 μg, 6 μg and 9 μg of Ile344Asn MTP. After 48 h, the media was replaced with fresh DMEM with 10% FBS and incubated overnight. The cells were collected, homogenized, and centrifuged at 12,000 rpm or 13,500 g for 10 min at 4°C. From the clear supernatant, 25 μg protein was separated on 8% SDS-PAGE and probed with anti-hMTP (A, top ), stripped, and probed with anti-β-actin antibodies for control (A, bottom ). For the TG transfer assay, 25 μg protein was used from the clear homogenate (B). Overnight-conditioned media was used to measure apoB-100 secretion by ELISA (C). For lipid transfer assays and MTP:PDI interaction studies, purified proteins were separated on SDS-PAGE and probed for anti-hMTP followed by anti-PDI antibody (D). Different aliquots of purified proteins were used to measure TG and PL transfer activity (E–F). The bars and error bars represent mean ± SD. To calculate the significance, one-way ANOVA nonparametric (multiple comparison) or two-way ANOVA was used. ∗∗∗ and ∗∗∗∗ represent P < 0.001 and P < 0.0001, respectively. The data are representative of three independent experiments performed with biological triplicates.

Article Snippet: Rabbit polyclonal anti-β-actin primary antibody (Cell Signaling #4967S) was used to detect the internal reference β-actin.

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Incubation, SDS Page, Control, Enzyme-linked Immunosorbent Assay, Purification, Comparison