rela/nfkb p65 [p ser276] antibody  (Bio-Techne corporation)

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Sustained activation of NF-κB through constitutively active IKKβ leads to senescence bypass in murine dermal fibroblasts.

doi: 10.1080/15384101.2024.2325802

Figure Lengend Snippet: Figure 2. Expression of constitutively active IKKβ bypasses cellular senescence. (a) Control (R26StopFLIKK2CA Cre-negative) and IKKβ-CA (R26StopFLIKK2CA Sm22αCre) skin fibroblasts were treated with MG132 (10 μM), a proteasome inhibitor, for 30 min prior to stimulation with IL-1β (10 ng/ml) for 5 min. Whole-cell lysates were analyzed by Western blotting using the indicated antibodies. Representative images are shown (n = 3). (b) Nuclear and cytoplasmic fractions were prepared from control and IKKβ-CA skin fibroblasts and analyzed by Western blotting. Nuclear p65 levels were quantified by ImageJ and normalized to the level of Lamin A (n = 3). (c)(d) Growth curves of control and IKKβ-CA skin fibroblasts under 20% and 3% oxygen conditions (n = 5). P2 Skin fibroblasts isolated from five adult control and IKKβ-CA mice were passaged in parallel under 20% oxygen conditions according to a 3T3 protocol and under 3% oxygen conditions according to a 3T1 protocol. The graph shows the cumulative number of cells in sequential passages. (e)(f) Representative images and quantification of senescence-associated β- galactosidase (SA-β-gal) staining of control and IKKβ-CA cells in passage 8 cultured under 3% and 20% oxygen conditions (n = 5). Scale bars, 100 µm. Error bars represent the standard error of the mean. (*p value < 0.05, **p value < 0.01)

Article Snippet: Phospho-p53 (Ser20) (# PA5–104741), phospho-p53 (Ser37) (#HYP80843) and phospho-RelA/p65 (Ser276) (#NB100–82086) antibodies were purchased from Invitrogen, MedChemExpress and Novus Biologicals, respectively.

Techniques: Expressing, Control, Western Blot, Isolation, Staining, Cell Culture

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Sustained activation of NF-κB through constitutively active IKKβ leads to senescence bypass in murine dermal fibroblasts.

doi: 10.1080/15384101.2024.2325802

Figure Lengend Snippet: Figure 3. Expression of nondegradable IκBα abolishes IKKβ-CA-induced senescence bypass. (a) Control (R26StopFLIKK2CA Cre-negative) and IKKβ-CA (R26StopFLIKK2CA Sm22αCre) skin fibroblasts were infected with lentivirus carrying an empty vector (EV) or FLAG-tagged IκBαSR. The expression of IκBαSR was confirmed by Western blotting using an antibody against the DYKDDDDK Tag. Representative images are shown (n = 4). (b) Nuclear and cytoplasmic fractions were prepared from control and IKKβ-CA skin fibroblasts harboring EV or IκBαSR. The nuclear p65 and p50 levels were measured by Western blotting, with quantification performed with ImageJ and normalized to the level of Lamin A (n = 4). (c)(d) Growth curves of control and IKKβ-CA skin fibroblasts harboring EV and IκBαSR cultured under 20% and 3% oxygen conditions, respectively. Skin fibroblasts were cultured under physiological oxygen level (3-5% oxygen) conditions until passage 3, at which time, they were infected with lentivirus. The cells were passaged again and treated with 2 μg/ml puromycin for 3 days under 3% oxygen conditions. The selected cells were passaged under 20% oxygen conditions according to a 3T2 protocol (c) or maintained under physiological oxygen level (3% oxygen) conditions according to a 3T1 protocol (d). The graph shows the cumulative number of cells in sequential passages. (e) Representative images and quantification of senescence-associated β-galactosidase (SA-β-gal) staining of control and IKKβ-CA cells harboring EV or IκBaSR cultured under 20% oxygen conditions. Scale bars, 100 µm. Error bars represent the standard error of the mean. (*p value < 0.05, **p value < 0.01)

Article Snippet: Phospho-p53 (Ser20) (# PA5–104741), phospho-p53 (Ser37) (#HYP80843) and phospho-RelA/p65 (Ser276) (#NB100–82086) antibodies were purchased from Invitrogen, MedChemExpress and Novus Biologicals, respectively.

Techniques: Expressing, Control, Infection, Plasmid Preparation, Western Blot, Cell Culture, Staining