Journal:

Article Title: RelA/p65 Regulation of I?B?

doi: 10.1128/MCB.25.12.4956-4968.2005

Figure Lengend Snippet: IκBβ is specifically regulated by p65 in fibroblasts. (A) Whole-cell extracts (WCEs) were prepared from immortalized MEFs either wild type or null for the p65 subunit of NF-κB, and the levels of the indicated proteins were analyzed by Western blotting. (B) Western blot assays to probe for IκBα and IκBβ in MEFs either wild type or null for various components in the NF-κB signaling pathway. Genotypes for all cell lines were confirmed by PCR analysis. (C) WCEs were prepared from vector or IκBα-SR C2C12 myoblasts, and Western blot assays were performed to probe for IκB proteins. (D) Primary MEFs were isolated from E13.5 embryos and genotyped for p65 by PCR. WCEs were prepared from p65 wild-type, heterozygous, and null MEFs, and Western blot assays were performed to probe for p65, IκBα, and IκBβ. α-Tubulin was used as a loading control. (E) Quantitation of IκB proteins from two Western blot assays performed with WCEs obtained from two independent litters as described for panel D. Levels of IκB proteins were normalized to α-tubulin and compared to the expression of IκB proteins obtained from wild-type cells, which was set to a value of 100%.

Article Snippet: Antibodies to IκBα (C21), IκBβ (C20, N20), Bcl-3, IKKγ, and p100 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); myosin heavy chain and α-tubulin were from Sigma (St. Louis, MO); p65 was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); and hemagglutinin (HA) was from Covance (Princeton, NJ).

Techniques: Western Blot, Plasmid Preparation, Isolation, Control, Quantitation Assay, Expressing

Journal:

Article Title: RelA/p65 Regulation of I?B?

doi: 10.1128/MCB.25.12.4956-4968.2005

Figure Lengend Snippet: p65 regulation of IκBβ is not limited to fibroblasts. (A) Histological hematoxylin-and-eosin (H&E) and immunohistochemical staining of p65 and IκBβ from longitudinal sections of p65+/+ (a, b, c) and p65−/− (d, e, f) embryos (×1 magnification). (B) Immunohistochemical staining of IκBβ of liver (a, d), lung (b, e), and brain (c, f) tissues from p65+/+ and p65−/− embryos (×4 magnification). (C) Primary fetal liver cells were prepared from E13.5 embryos, and after genotypes were confirmed, Western blot assays were performed to probe for p65, IκBα, and IκBβ. α-Tubulin was used as a loading control. (D) p65 and IκBβ immunohistochemical staining of heart tissue from p65+/+ and p65−/− embryos (×4 magnification). (E) p65 and IκBβ immunohistochemical staining of forelimbs from p65+/+ and p65−/− embryos. To confirm skeletal muscle staining, serial sections of forelimbs were separately stained for myosin heavy chain (MyHC; arrowheads denote muscle tracks; ×4 magnification).

Article Snippet: Antibodies to IκBα (C21), IκBβ (C20, N20), Bcl-3, IKKγ, and p100 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); myosin heavy chain and α-tubulin were from Sigma (St. Louis, MO); p65 was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); and hemagglutinin (HA) was from Covance (Princeton, NJ).

Techniques: Immunohistochemical staining, Staining, Western Blot, Control

Journal:

Article Title: RelA/p65 Regulation of I?B?

doi: 10.1128/MCB.25.12.4956-4968.2005

Figure Lengend Snippet: p65 regulates various forms of IκBβ in postnatal development in a tissue-specific manner. p65+/− TNF-α−/− mice were bred to generate p65+/+ TNF-α−/− and p65−/− TNF-α−/− progeny. (A) At 4 weeks of age, mice were sacrificed and tissue homogenates were prepared (skmc, skeletal muscle). Western blot assays were then performed to probe for p65, IκBα, IκBβ, and α-tubulin. Each Western blot assay is representative of a total of four different blot assays derived from two sets of littermates. (B) Western blot assay to probe for IκBβ in p65+/+ TNF-α−/− brain, skin, skeletal muscle, and heart tissues. Arrows denote IκBβ forms I through IV. (C) Similar extracts as in panel A were either left untreated or treated with phosphatase (PPase) enzyme, and Western blot assay was performed to probe for IκBβ. Arrows denote phosphorylated forms of IκBβ, and asterisks denote the shifted (dephosphorylated) forms of IκBβ. (D) The N-terminal IκBβ antibody (N20) was used in a Western analysis to verify IκBβ forms in p65+/+ TNF-α−/− tissues.

Article Snippet: Antibodies to IκBα (C21), IκBβ (C20, N20), Bcl-3, IKKγ, and p100 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); myosin heavy chain and α-tubulin were from Sigma (St. Louis, MO); p65 was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); and hemagglutinin (HA) was from Covance (Princeton, NJ).

Techniques: Western Blot, Derivative Assay

Journal:

Article Title: RelA/p65 Regulation of I?B?

doi: 10.1128/MCB.25.12.4956-4968.2005

Figure Lengend Snippet: IκBβ downregulation in p65−/− MEFs is regulated by the proteasome independent of classical IKK signaling. (A) MEFs wild type and null for p65 were treated with TNF-α, and at indicated time points a real-time PCR was used to measure IκBα and IκBβ RNAs. (B) p65+/+ and p65−/− MEFs were incubated for 2 h in methionine- and cysteine-free DMEM and subsequently labeled with a [35S]methionine/cysteine mix for an additional hour. Whole-cell extracts were prepared, and IκBβ was immunoprecipitated either with IgG (control) or with an IκBβ-specific antibody. Complexes were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by exposing dried gels to film for up to 3 days (arrow denotes IκBβ). (C) p65+/+ and p65−/− MEFs were treated with cycloheximide (CHX; 10 μg/ml), and at indicated times whole-cell extracts were prepared and Western blot assays performed to probe for IκBβ. Levels of IκBβ were quantitated from an average of three Western blot assays. (D) Similar conditions were used to treat p65+/+ and p65−/− MEFs with MG-132, ALLN, and lactacysteine (all at 10 μM). (E) Kinase assays (KA) were performed with IKKγ immunoprecipitates from p65+/+ and p65−/− MEFs incubated with glutathione S-transferase-IκBα as a substrate. Whole-cell extracts from p65+/+ and p65−/− MEFs used in kinase assay were analyzed by Western bloting to probe for IKKα and p65. (F) Western blot assays to probe for IκBβ and tubulin in p65−/− MEFs transfected with vector control or catalytically inactive IKKα or IKKβ proteins (top). Inhibitory activity of IKK proteins was verified by luciferase NF-κB reporter assays (bottom). (G) p65−/− MEFs were transfected with either HA-tagged wild-type (WT) IκBβ or HA-tagged IκBβ mutated at serines 19 and 23 to either alanine (S19/23A) or glutamic acid (S19/23E) residues. After 24 h, cells were treated with cycloheximide (10 μg/ml) for the indicated times. Whole-cell extracts were then prepared and Western blot assays performed to probe for HA and α-tubulin.

Article Snippet: Antibodies to IκBα (C21), IκBβ (C20, N20), Bcl-3, IKKγ, and p100 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); myosin heavy chain and α-tubulin were from Sigma (St. Louis, MO); p65 was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); and hemagglutinin (HA) was from Covance (Princeton, NJ).

Techniques: Real-time Polymerase Chain Reaction, Incubation, Labeling, Immunoprecipitation, Control, Polyacrylamide Gel Electrophoresis, Western Blot, Kinase Assay, Transfection, Plasmid Preparation, Activity Assay, Luciferase

Journal:

Article Title: RelA/p65 Regulation of I?B?

doi: 10.1128/MCB.25.12.4956-4968.2005

Figure Lengend Snippet: MEFs stably expressing IκBβ exhibit a growth defect and increased apoptosis. p65−/− MEFs were infected with either a pBabe vector retrovirus or a virus expressing a degradation-resistant form of IκBβ tagged with an HA epitope (pBabeIκBβ-SR). (A) Extracts from p65−/−-expressing vector or HA-IκBβ-SR cells were prepared, and Western blot assays were performed to probe for HA and α-tubulin. (B) p65−/− cells expressing either the vector control or IκBβ-SR were treated with TNF-α, and at the indicated times nuclear extracts were prepared for EMSA. A supershift EMSA was performed with antibodies against p50, p65, or c-Rel to confirm the composition of NF-κB complexes (arrowheads denote supershifted subunits). (C) p65−/− MEFs were infected with a vector control or IκBβ-SR-expressing virus. Following 4 days of cell expansion under 1-μg puromycin selection, the cell number was determined in both cell lines. Cell number was normalized to vector control cells, which was set to a value of 100% growing cells. (D) Growth curves of p65+/+ or p65−/− cells expressing either a vector control or IκBβ-SR. (E) Growth curves identical to those performed in panel B from p65−/− cells infected with a vector control, IκBα-SR, or IκBβ-SR pBabe retrovirus. Cell number was normalized to the vector, which was set to a value of 100%. (F) Cells infected as in panel E were expanded under puromycin selection for 2 days and subsequently stained for Annexin V.

Article Snippet: Antibodies to IκBα (C21), IκBβ (C20, N20), Bcl-3, IKKγ, and p100 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); myosin heavy chain and α-tubulin were from Sigma (St. Louis, MO); p65 was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); and hemagglutinin (HA) was from Covance (Princeton, NJ).

Techniques: Stable Transfection, Expressing, Infection, Plasmid Preparation, Virus, Western Blot, Control, Selection, Staining

Journal: medRxiv

Article Title: The angiotensin type 2 receptor agonist C21 restores respiratory function in COVID19 - a double-blind, randomized, placebo-controlled Phase 2 trial

doi: 10.1101/2021.01.26.21250511

Figure Lengend Snippet: Daily estimates of the proportion of patients in need for supplemental oxygen therapy. The left panel shows the total intention to treat population (C21 N=51, placebo N=55). The upper right panel shows patients in need for supplemental oxygen on the day of randomization (C21 N=37, placebo N=39), and the lower right panel shows patients not in need for oxygen on the day of randomization (C21 N=14, placebo N=16).

Article Snippet: In conclusion, C21 on top of standard of care, including glucocorticoids and remdesivir, significantly improved respiratory function reflected by a reduced need for supplemental oxygen in hospitalized COVID-19 patients.

Techniques: