Journal: American journal of respiratory cell and molecular biology

Article Title: Secretion of interleukin-1 receptor antagonist from human mast cells after immunoglobulin E-mediated activation and after segmental antigen challenge.

doi: 10.1165/ajrcmb.25.6.4541

Figure Lengend Snippet: Figure 1. Time course of mRNA expression of IL-1 cyto- kines and receptors produced by cultured huMCs after ag- gregation of FcεRI. Cells were exposed to anti–NP-IgE over- night, followed by the addition of antigen () to paired cell cultures. Total RNA was isolated at the indicated times and the RPA was performed. A representative result is shown (A). To establish the identity of each protected fragment, the known size and migration distance of the unprotected probe was used to prepare a standard curve. Human control RNA (BD Pharmingen) was used as a positive control and yeast tRNA was used as a negative control. Semiquantification of IL-1ra (B), IL-1RI (C), and IL-1 (D) expression in huMCs before (open bars) or after (filled bars) aggregation of FcεRI was performed by measuring the band density of the relative expression of each mRNA with respect to L32 mRNA after each background was subtracted. IL-1RII and IL-1 mRNAs were below detection levels. For IL-1ra and IL-1 the results were repeated in five separate experiments through 8 h using cells cultured from different donors. These data are pre- sented as means SEM; *P 0.05, **P 0.005, ***P 0.001 compared with 0 time. The 4-h error bar in activated cells in D was too small to diagram.

Article Snippet: The following antibodies were applied for 30 min at previously titrated optimal dilutions: goat antihuman IL-1ra (R&D Systems), AA1 to mast-cell tryptase (Dako Corp., Carpinteria, CA), or goat IgG or mouse IgG 1 control (Dako Corp.).

Techniques: Expressing, Produced, Cell Culture, Isolation, Migration, Control, Positive Control, Negative Control

Journal: American journal of respiratory cell and molecular biology

Article Title: Secretion of interleukin-1 receptor antagonist from human mast cells after immunoglobulin E-mediated activation and after segmental antigen challenge.

doi: 10.1165/ajrcmb.25.6.4541

Figure Lengend Snippet: Figure 4. Inhibition of IL-1–induced IL-8 production by A549 epithelial cells by IL-1ra or mast-cell lysates. A549 cells were in- cubated with IL-1 at a concentration of 50 ng/ml for 16 h in the presence or absence of either rhIL-1ra (A) or huMC lysate (B). Cell-free supernates were collected and assayed for IL-8 by ELISA. The increase was downregulated by (A) increasing con- centrations of standard IL-1ra, and (B) increasing concentrations of huMC lysate.

Article Snippet: The following antibodies were applied for 30 min at previously titrated optimal dilutions: goat antihuman IL-1ra (R&D Systems), AA1 to mast-cell tryptase (Dako Corp., Carpinteria, CA), or goat IgG or mouse IgG 1 control (Dako Corp.).

Techniques: Inhibition, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: American journal of respiratory cell and molecular biology

Article Title: Secretion of interleukin-1 receptor antagonist from human mast cells after immunoglobulin E-mediated activation and after segmental antigen challenge.

doi: 10.1165/ajrcmb.25.6.4541

Figure Lengend Snippet: Figure 3. Time course of IL-1ra release from cultured huMCs af- ter FcεRI aggregation. huMCs were incubated with anti–NP-IgE overnight at 37C and washed, and NP-BSA was added to aggre- gate FcεRI (time 0). Plates were then centrifuged at the indicated time points, the supernates removed, and cells lysed. IL-1ra was measured by ELISA in both huMC lysates (open bars) and su- pernates (filled bars) of mast cells (*P 0.05, and **P 0.005 compared with time 0). Data presented are the results of three experiments performed on huMCs cultured from a single donor.

Article Snippet: The following antibodies were applied for 30 min at previously titrated optimal dilutions: goat antihuman IL-1ra (R&D Systems), AA1 to mast-cell tryptase (Dako Corp., Carpinteria, CA), or goat IgG or mouse IgG 1 control (Dako Corp.).

Techniques: Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay

Journal: American journal of respiratory cell and molecular biology

Article Title: Secretion of interleukin-1 receptor antagonist from human mast cells after immunoglobulin E-mediated activation and after segmental antigen challenge.

doi: 10.1165/ajrcmb.25.6.4541

Figure Lengend Snippet: Figure 2. IL-1ra protein in huMCs and in supernates from huMCs after aggregation of FcεRI. (A) After staining of permeabilized cells with a phycoerythrin-conjugated anti–IL-1ra mAb (thick line) or an isotype control mouse IgG1 (thin line), FACS analysis was per- formed to detect the intracellular expression of IL-1ra. (B) huMCs were incubated with anti–NP-IgE overnight at 37C and washed, and NP-BSA was added to aggregate FcεRI. Plates were then centrifuged and the supernates removed. IL-1ra was measured by ELISA in supernates of stimulated (filled bar) and unstimulated (open bar) cells at 20 h (n 4; with cells for each experiment cul- tured from separate donors). (C) Western blot analysis of IL-1ra in huMC supernatants and lysates. ST, rhIL-1ra standard; Lys, concentrated lysate; Supp, concentrated supernate; Lys HMC-1, concentrated HMC-1 lysate. These samples were subjected to electrophoresis on 4 to 12% Bis-Tris gel under reducing condi- tions. After electrophoresis, proteins were blotted and incubated with goat antihuman IL-1ra.

Article Snippet: The following antibodies were applied for 30 min at previously titrated optimal dilutions: goat antihuman IL-1ra (R&D Systems), AA1 to mast-cell tryptase (Dako Corp., Carpinteria, CA), or goat IgG or mouse IgG 1 control (Dako Corp.).

Techniques: Staining, Control, Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot, Electrophoresis

Journal: American journal of respiratory cell and molecular biology

Article Title: Secretion of interleukin-1 receptor antagonist from human mast cells after immunoglobulin E-mediated activation and after segmental antigen challenge.

doi: 10.1165/ajrcmb.25.6.4541

Figure Lengend Snippet: Figure 5. IL-1ra in human lung mast cells. (A) Sequential 2-m sections of a surgically resected lung immuno- stained for tryptase (i) and IL-1ra (ii), demonstrating colocalization of IL-1ra to tryptase mast cells (filled arrows). Open arrows indicate IL- 1ra tryptase cells. (B) IL-1ra pro- duction from human lung mast cells (106 cells/ml) stimulated with FcεRI aggregation. Purified human lung– derived mast cells were incubated with anti–NP-IgE overnight at 37C and washed, and NP-BSA was added to aggregate FcεRI. Plates were then centrifuged and the supernates re- moved. IL-1ra was measured by ELISA in supernates of stimulated (filled bar) and unstimulated (open bar) cells at 20 h (n 2).

Article Snippet: The following antibodies were applied for 30 min at previously titrated optimal dilutions: goat antihuman IL-1ra (R&D Systems), AA1 to mast-cell tryptase (Dako Corp., Carpinteria, CA), or goat IgG or mouse IgG 1 control (Dako Corp.).

Techniques: Staining, Purification, Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: American journal of respiratory cell and molecular biology

Article Title: Secretion of interleukin-1 receptor antagonist from human mast cells after immunoglobulin E-mediated activation and after segmental antigen challenge.

doi: 10.1165/ajrcmb.25.6.4541

Figure Lengend Snippet: Figure 6. IL-1ra levels and isoforms in BALF from seg- mental antigen challenge. Segmental antigen challenge was performed as described in MATERIALS AND METHODS, with (A) before- (Pre) and after- (Post) antigen challenge eosin- ophil number (** indicates a significant difference between eosinophil number in BALF after antigen challenge when compared with before challenge; P 0.005; (B) before- and after-challenge total cell number (* indicates a signifi- cant difference between total cell number in BALF after antigen challenge when compared with before challenge; P 0.05); and (C) IL-1ra (filled bars) and tryptase (open bars) found in cell-free supernates from BALF. Normal subjects (Normal) were 10 patients who were nonatopic and nonasthmatic. There was a significant difference (*P 0.05) between IL-1ra levels in BALF after antigen chal- lenge when compared with before-challenge and normal lev- els. (D) Western blotting of BALF shows the 17-kD form as prominent in BALF with a minimal band at 22 kD. ST, rhIL-1ra 17-kD standard; BAL 1 and 2 are representative samples of after-challenge BALF.

Article Snippet: The following antibodies were applied for 30 min at previously titrated optimal dilutions: goat antihuman IL-1ra (R&D Systems), AA1 to mast-cell tryptase (Dako Corp., Carpinteria, CA), or goat IgG or mouse IgG 1 control (Dako Corp.).

Techniques: Western Blot

Journal: Frontiers in Pharmacology

Article Title: Shengma Biejia Decoction Inhibits Cell Growth in Multiple Myeloma by Inducing Autophagy-Mediated Apoptosis Through the ERK/mTOR Pathway

doi: 10.3389/fphar.2021.585286

Figure Lengend Snippet: SMBJD is safe and could inhibit the expression of autophagy-related proteins, promote apoptosis-related proteins in vivo . (A) Representative pictures of peer group mouse liver, spleen, and kidney after HE staining (× 200), scale bar = 100 µm. (B) The expression levels of P62, LC3, and cleaved caspase-3 in transplanted tumors were detected using immunohistochemistry (× 200), scale bar = 100 µm. (C) Extracts from transplanted tumors were analyzed for autophagy-related and apoptosis-related proteins expression by western blot. (D) The corresponding expression of P62, LC3, and cleaved caspase-3 levels in tumor tissue are shown as histograms. (E) The corresponding autophagy-related and apoptosis-related proteins expression by western blot are shown as histograms. (* p < 0.05 compared with the model group).

Article Snippet: Sections were then blocked with 3% bovine serum albumin for 30 min and incubated with rabbit monoclonal primary antibodies against P62 (1:20–1:200, Proteintech, IL, United States), LC3 (1:50–500, Proteintech, IL, United States), cleaved caspase-3 (1:200, CST, MA, United States), ERK1/2 (1:400–1,600, CST, MA, United States), phosphorylated ERK1/2 (1:400, CST, MA, United States), Ser2448 mTOR (1:100, CST, MA, United States), and phosphorylated Ser2448 mTOR (1:100, CST, MA, United States) overnight at 4°C.

Techniques: Expressing, In Vivo, Staining, Immunohistochemistry, Western Blot

Journal: International journal of molecular sciences

Article Title: Readdressing the Localization of Apolipoprotein E (APOE) in Mitochondria-Associated Endoplasmic Reticulum (ER) Membranes (MAMs): An Investigation of the Hepatic Protein-Protein Interactions of APOE with the Mitochondrial Proteins Lon Protease (LONP1), Mitochondrial Import Receptor Subunit TOM40 (TOMM40) and Voltage-Dependent Anion-Selective Channel 1 (VDAC1).

doi: 10.3390/ijms251910597

Figure Lengend Snippet: Figure 1. APOE accumulates in the MAMs of cultured hepatocytes, equally evident during APOE3 and APOE4 overexpression: (a) subcellular fractions were isolated from Huh7 cells by Percoll density gradient ultracentrifugation and analyzed by Western blotting. Representative images show the accumulation of the APOE protein in pure MAMs. Respective marker proteins were detected to visualize the purity of mitochondria (COX), MAMs (CANX) and cytosol (TUB); (b) APOE protein levels were quantified in the whole cell sample and crude and pure MAM fractions and normalized to the APOE level in the whole cell sample. In the pure MAM, the APOE protein level was significantly higher compared to the whole cell (p < 0.05, unpaired t-test) as indicated by an asterisk (*). The data are means ± SEM (n = 3); (c) no APOE isoform-dependent difference was observed in the accumulation of APOE in the pure MAM fraction in APOE3- and APOE4-transfected Huh7 cells. Data are means ± SEM (n = 2) and the accumulation of APOE in MAMs is related to the APOE protein level in the whole cell samples; subcellular fractions: c. mito., crude mitochondria; p. mito., pure mitochondria; c. MAM, crude mitochondria ER-membranes (MAMs), p. MAM, pure MAM.

Article Snippet: Then, the cells were incubated with ligase (30 min, 37 ◦C), polymerase (100 min, 37 ◦C) and fluorescent anti-APOE antibody (1:20, 90 min, room temperature; sc-13521 AF546, Santa Cruz, Dallas, TX, USA).

Techniques: Cell Culture, Over Expression, Isolation, Western Blot, Marker, Transfection

Journal: International journal of molecular sciences

Article Title: Readdressing the Localization of Apolipoprotein E (APOE) in Mitochondria-Associated Endoplasmic Reticulum (ER) Membranes (MAMs): An Investigation of the Hepatic Protein-Protein Interactions of APOE with the Mitochondrial Proteins Lon Protease (LONP1), Mitochondrial Import Receptor Subunit TOM40 (TOMM40) and Voltage-Dependent Anion-Selective Channel 1 (VDAC1).

doi: 10.3390/ijms251910597

Figure Lengend Snippet: Figure 2. The number of visualized MERCs in cultured hepatocytes and MAM-assembling protein levels in the mouse liver are similar in presence of APOE3 and APOE4: (a) representative images of MERC-PLA experiments in APOE-transfected Huh7 cells that were incubated with 1 or 5 g/L glucose for six hours. VDAC1-IP3R1 PLA signals are shown in green, APOE was additionally stained in red to identify successfully transfected cells, and cell nuclei appeared blue by staining with DAPI. 400× magnification; scale bar 5 µm; (b) PLA signals from at least 100 cells per sample from three independent experiments were quantified showing a significant reduction of PLA signals per cell in all samples in response to the glucose challenge. No difference was observed comparing APOE3-, APOE4- and mock- transfected cells. Significance was accepted at p < 0.05, indicated with an asterisk (*); a two-way ANOVA was performed followed by the Šídák’s multiple comparisons test; (c) MAM-assembling as well as ER and mitochondrial marker proteins were analyzed in the livers of APOE-targeted replacement mice by Western blotting and representative images are shown; (d) densitometric analysis revealed no significant differences between APOE3 and APOE4 mice (unpaired t-test). Target band intensity was normalized by total protein load and related to the mean of APOE3 mice. Data are means ± SEM (n = 5–6).

Article Snippet: Then, the cells were incubated with ligase (30 min, 37 ◦C), polymerase (100 min, 37 ◦C) and fluorescent anti-APOE antibody (1:20, 90 min, room temperature; sc-13521 AF546, Santa Cruz, Dallas, TX, USA).

Techniques: Cell Culture, Transfection, Incubation, Staining, Marker, Western Blot

Journal: International journal of molecular sciences

Article Title: Readdressing the Localization of Apolipoprotein E (APOE) in Mitochondria-Associated Endoplasmic Reticulum (ER) Membranes (MAMs): An Investigation of the Hepatic Protein-Protein Interactions of APOE with the Mitochondrial Proteins Lon Protease (LONP1), Mitochondrial Import Receptor Subunit TOM40 (TOMM40) and Voltage-Dependent Anion-Selective Channel 1 (VDAC1).

doi: 10.3390/ijms251910597

Figure Lengend Snippet: Figure 3. Mitochondrial APOE-interacting proteins are remotely connected to MAMs, with no apparent difference comparing APOE3- and APOE4-transfected cells. (a) Visualization of the eleven higher abundant protein–protein interactions (PPIs) shared by APOE3- and APOE4-transfected cells compared to unmodified Huh7 cells. PPIs were categorized by their main cellular localization identified according to The Human Gene Database GeneCards. Hitherto unknown PPIs (7/11) are highlighted by the greater thickness and blue color of the borderline. (b) Representative Western blot images of the presence of the APOE-interacting proteins LONP1, TOMM40 and VDAC1 in subcellular fractions isolated from Huh7 cells. Respective marker proteins for mitochondrial and ER-related MAM proteins were detected (GRP75, mitochondria, MAM; and PEMT, ER and MAM). (c) Representative Western blot images showing APOE, LONP1, TOMM40 and VDAC1 as well as the ER/MAM marker CANX in subcellular fractions of APOE3- and APOE4-transfected cells. (d) Target band intensities were quantified and normalized by total protein load per lane. The target protein level in the pure MAM fraction was related to the whole cell sample showing similar results in APOE3- and APOE4-transfected cells. Data are means ± SEM (n = 2). Subcellular fractions: c. mito., crude mitochondria; p. mito., pure mitochondria; c. MAM, crude mitochondria ER membranes (MAMs), p. MAM, pure MAM.

Article Snippet: Then, the cells were incubated with ligase (30 min, 37 ◦C), polymerase (100 min, 37 ◦C) and fluorescent anti-APOE antibody (1:20, 90 min, room temperature; sc-13521 AF546, Santa Cruz, Dallas, TX, USA).

Techniques: Transfection, Protein-Protein interactions, Western Blot, Isolation, Marker

Journal: International journal of molecular sciences

Article Title: Readdressing the Localization of Apolipoprotein E (APOE) in Mitochondria-Associated Endoplasmic Reticulum (ER) Membranes (MAMs): An Investigation of the Hepatic Protein-Protein Interactions of APOE with the Mitochondrial Proteins Lon Protease (LONP1), Mitochondrial Import Receptor Subunit TOM40 (TOMM40) and Voltage-Dependent Anion-Selective Channel 1 (VDAC1).

doi: 10.3390/ijms251910597

Figure Lengend Snippet: Figure 4. Presence in MAMs and extent of the PPI of selected candidates and APOE in ER-stressed cultured hepatocytes. (a) Western blot images of LONP1, TOMM40, VDAC1 and APOE detection in subcellular fractions of unmodified Huh7 cells. Thapsigargin treatment (50 µM, 24 h) provoked the accumulation of LONP1 and APOE in MAMs relative to the whole cell sample and compared to untreated control cells. GRP75 served as a marker for MAMs and the positive control for thapsigargin induced MAM protein translocation. c. MAM, crude mitochondria ER membranes (MAMs), p. MAM, pure MAM; (b) representative Western blot images of LONP1, TOMM40 and VADC1 in APOE co-IP samples from unmodified Huh7 cells treated with thapsigargin (50 µM, 24 h). No signals were visible in the IP negative control (no antibody used, no AB). S, IP supernatant, inp., input control; (c) target band intensity was normalized by the corresponding APOE band intensity. Relative target protein levels in thapsigargin-stressed cells were related to the mean of untreated cells (expressed as thapsigargin-induced change). Data are means from three individual cell culture experiments and co-IP (n = 3).

Article Snippet: Then, the cells were incubated with ligase (30 min, 37 ◦C), polymerase (100 min, 37 ◦C) and fluorescent anti-APOE antibody (1:20, 90 min, room temperature; sc-13521 AF546, Santa Cruz, Dallas, TX, USA).

Techniques: Cell Culture, Western Blot, Control, Marker, Positive Control, Translocation Assay, Co-Immunoprecipitation Assay, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly *

doi: 10.1074/jbc.M114.591545

Figure Lengend Snippet: Phosphorylation of TPX2 at Thr72 is inhibited by the Cdk inhibitor roscovitine. A, the levels of Thr(P)72 were reduced by roscovitine treatment. HeLa cells were first treated with 100 ng/ml of nocodazole for 16 h and then once synchronized, treated with dimethyl sulfoxide (as a control), or 20 and 40 μm roscovitine for 30 min. Cells were harvested, lysed, and TPX2 was immunoprecipitated with pan-TPX2 Abs (clone 184). SDS-PAGE was performed and followed by Western blotting with Thr(P)72 and pan-TPX2 Abs (clone 184). B, cyclin B1 levels were also used to confirm that roscovitine-treated cells had remained in mitosis. The levels of p-Cdk/MAPK substrates (PX(S*/T*)P or (S*/T*)PX(R/K) motif) were also used to confirm the effectiveness of the treatment. Actin levels were used as loading control.

Article Snippet: Antibodies Primary Abs against Cdk1, Cdk2 (Santa Cruz), Thr(P) 72 TPX2 (homemade, described above), Cyclin B (Abcam), α-actin (Chemicon), TPX2 (clone 183, epitope: 150–200 amino acids of human TPX2, Novus Biologicals; clone 184, epitope: 700–749 amino acids of TPX2, Novus Biologicals), phospho-Cdk/MAPK substrates (34B2, Cell Signaling), Cy3 conjugated-β-tubulin (Sigma), p-Aurora A (ab18318, Abcam), and monoclonal TPX2 (18D5-1, Abcam) were used for Western blots, immunoprecipitations, and/or immunofluorescence experiments.

Techniques: Immunoprecipitation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Knockdown of the E3 Ubiquitin ligase UBR5 and its role in skeletal muscle anabolism

doi: 10.1101/2020.06.05.116145

Figure Lengend Snippet: (A) Total RNA concentration (assessed as µg/mg of muscle) was significantly reduced ( P = 0.01) in the UBR5 RNAi muscles. (B & C) RNAi transfected muscles also displayed a significant reduction (∼17%, P = 0.05) in muscle protein synthesis (B) assessed via western blots for puromycin incorporation (C). (D) Western blot images for the ERK signaling pathway. (E) Decreased phosphorylation levels of ERK1/2 (p44/42 MAPK) (P = 0.03) and (F) increased phosphorylation levels of p90RSK ( P = 0.04) with UBR5 RNAi. (G) Reduced levels of total p90RSK ( P = 0.02). Data presented as Mean ± SEM. Total protein loading of the membranes captured from images using stain-free gel technology was used as the normalization control for all blots. N = 5 per group. Statistical significance is depicted where present (P ≥ 0.05).

Article Snippet: The following primary antibodies were used all used at a concentration of 1:1000: Cell Signaling Technologies (Danvers, MA, USA) – phospho-ERK1/2 Thr202/Tyr204 (Cat# 4370, RRID:AB_2315112), ERK1/2 (Cat# 4695, RRID:AB_390779), phospho-p90RSK Ser380 (Cat# 11989, RRID:AB_2687613), p90RSK (Cat# 9355, RRID:AB_659900), phospho-MSK1 Thr581 (Cat# 9595, RRID:AB_2181783), phospho-MEK1/2 Ser217/221 (Cat# 9154, RRID:AB_2138017), phospho-Akt Ser473 (Cat# 4060, RRID:AB_2315049), Akt (Cat# 9272, RRID:AB_329827), phospho-GSK3β Ser9 (cat. Cat# 9336, RRID:AB_331405), GSK3β (Cat# 9332, RRID:AB_2335664), phospho-p70S6K Thr 389 (Cat# 9205, RRID:AB_330944), p70S6K (Cat# 9202, RRID:AB_331676), phospho-rpS6 Ser240/244 (Cat# 5364, RRID:AB_10694233), rpS6 (Cat# 2217, RRID:AB_331355), phospho-4EBP1 Thr37/46 (Cat# 2855, RRID: AB_560835), 4EBP1 (Cat# 9644, RRID:AB_2097841) UBR5 (Cat# 65344, RRID:AB_2799679), PP2Ac (Cat# 2259, RRID:AB_561239), eIF4e (Cat# 9742, RRID:AB_823488) and EMD Millipore – puromycin (Cat# MABE343, RRID:AB_2566826).

Techniques: Concentration Assay, Transfection, Western Blot, Staining

Journal: bioRxiv

Article Title: Knockdown of the E3 Ubiquitin ligase UBR5 and its role in skeletal muscle anabolism

doi: 10.1101/2020.06.05.116145

Figure Lengend Snippet: (A) Phosphorylated p90RSK, (B) total p90RSK, (C) Phosphorylated ERK1/2 and (D) Total ERK. All n = 5 per group – EV control and UBR5 RNAi. Data presented as Mean ± SEM. Total protein loading of the membranes captured from images using stain-free gel technology was used as the normalization control for all blots. Statistical significance is depicted where present (P ≥ 0.05).

Article Snippet: The following primary antibodies were used all used at a concentration of 1:1000: Cell Signaling Technologies (Danvers, MA, USA) – phospho-ERK1/2 Thr202/Tyr204 (Cat# 4370, RRID:AB_2315112), ERK1/2 (Cat# 4695, RRID:AB_390779), phospho-p90RSK Ser380 (Cat# 11989, RRID:AB_2687613), p90RSK (Cat# 9355, RRID:AB_659900), phospho-MSK1 Thr581 (Cat# 9595, RRID:AB_2181783), phospho-MEK1/2 Ser217/221 (Cat# 9154, RRID:AB_2138017), phospho-Akt Ser473 (Cat# 4060, RRID:AB_2315049), Akt (Cat# 9272, RRID:AB_329827), phospho-GSK3β Ser9 (cat. Cat# 9336, RRID:AB_331405), GSK3β (Cat# 9332, RRID:AB_2335664), phospho-p70S6K Thr 389 (Cat# 9205, RRID:AB_330944), p70S6K (Cat# 9202, RRID:AB_331676), phospho-rpS6 Ser240/244 (Cat# 5364, RRID:AB_10694233), rpS6 (Cat# 2217, RRID:AB_331355), phospho-4EBP1 Thr37/46 (Cat# 2855, RRID: AB_560835), 4EBP1 (Cat# 9644, RRID:AB_2097841) UBR5 (Cat# 65344, RRID:AB_2799679), PP2Ac (Cat# 2259, RRID:AB_561239), eIF4e (Cat# 9742, RRID:AB_823488) and EMD Millipore – puromycin (Cat# MABE343, RRID:AB_2566826).

Techniques: Staining

Journal: bioRxiv

Article Title: Knockdown of the E3 Ubiquitin ligase UBR5 and its role in skeletal muscle anabolism

doi: 10.1101/2020.06.05.116145

Figure Lengend Snippet: UBR5 knockdown (X) increases phosphatase PP2Ac, reduces ERK and Akt phosphorylation (RED), reduces total p90RSK and eIF4e (PURPLE), chronically increases the phosphorylation of p70S6K and rpS6 (GREEN).

Article Snippet: The following primary antibodies were used all used at a concentration of 1:1000: Cell Signaling Technologies (Danvers, MA, USA) – phospho-ERK1/2 Thr202/Tyr204 (Cat# 4370, RRID:AB_2315112), ERK1/2 (Cat# 4695, RRID:AB_390779), phospho-p90RSK Ser380 (Cat# 11989, RRID:AB_2687613), p90RSK (Cat# 9355, RRID:AB_659900), phospho-MSK1 Thr581 (Cat# 9595, RRID:AB_2181783), phospho-MEK1/2 Ser217/221 (Cat# 9154, RRID:AB_2138017), phospho-Akt Ser473 (Cat# 4060, RRID:AB_2315049), Akt (Cat# 9272, RRID:AB_329827), phospho-GSK3β Ser9 (cat. Cat# 9336, RRID:AB_331405), GSK3β (Cat# 9332, RRID:AB_2335664), phospho-p70S6K Thr 389 (Cat# 9205, RRID:AB_330944), p70S6K (Cat# 9202, RRID:AB_331676), phospho-rpS6 Ser240/244 (Cat# 5364, RRID:AB_10694233), rpS6 (Cat# 2217, RRID:AB_331355), phospho-4EBP1 Thr37/46 (Cat# 2855, RRID: AB_560835), 4EBP1 (Cat# 9644, RRID:AB_2097841) UBR5 (Cat# 65344, RRID:AB_2799679), PP2Ac (Cat# 2259, RRID:AB_561239), eIF4e (Cat# 9742, RRID:AB_823488) and EMD Millipore – puromycin (Cat# MABE343, RRID:AB_2566826).

Techniques:

Journal:

Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

doi: 10.1128/MCB.22.22.7929-7941.2002

Figure Lengend Snippet: GILZ overexpression inhibits ERK-1/2, MEK-1/2, and Raf-1 but not JNK phosphorylation. Clones transfected with pcDNA3 (PV6) or pcDNA3-GILZ (GIRL-19) were stimulated for 5 or 60 min with plastic-bound anti-CD3 MAb. Whole-cell lysates were probed with an antibody specific for phosphorylated ERK-1/2 (pERK-1/2) (A), MEK-1/2 (pMEK) (B), or Raf-1 (pRaf-1) (C). Western blots were also performed with anti-ERK-1/2, anti-MEK-1/2, or anti-Raf-1 antibodies to verify that no modulation of protein expression occurred or with β-tubulin to verify that an equivalent amount of proteins was loaded in each lane. PV6 or GIRL-19 was stimulated for the times indicated with plastic-bound anti-CD3 MAb. (D) Whole-cell lysates were probed with an antibody recognizing both phosphorylated forms of JNK: p54 and p46 (pSAPK/JNK). C, untreated cells.

Article Snippet: The primary antibodies were a polyclonal rabbit antiserum recognizing GILZ, a polyclonal rabbit anti-mouse phospho-ERK-1/2, and anti-mouse ERK-1/2 (Cell Signaling), anti-Fos, anti-Jun, anti-Raf-1, monoclonal rat anti-mouse phospho-Raf-1 (series 338; Upstate Biotechnology), anti-MEK, and polyclonal rabbit anti-human phospho-MEK (Ser 217/221; Cell Signaling) antibodies.

Techniques: Over Expression, Clone Assay, Transfection, Western Blot, Expressing

Journal:

Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

doi: 10.1128/MCB.22.22.7929-7941.2002

Figure Lengend Snippet: GILZ interacts with endogenous Raf-1 in mouse thymocytes. Mouse thymocytes were treated for 6 h with DEX (100 nM), and cell lysates were incubated with GST or GST-GILZ beads. Binding of Raf-1 (A), MEK-1/2 (B), and ERK-1/2 (C) was visualized by Western blotting. Whole-cell lysates from thymocytes left untreated or treated with DEX were immunoprecipitated with an anti-Raf-1 or control isotype antibody (4 μg/500 μg of protein). (D and E) Nitrocellulose membrane was probed with an anti-GILZ antiserum (D) and then stripped and reprobed with anti-Raf-1 antibody (E).

Article Snippet: The primary antibodies were a polyclonal rabbit antiserum recognizing GILZ, a polyclonal rabbit anti-mouse phospho-ERK-1/2, and anti-mouse ERK-1/2 (Cell Signaling), anti-Fos, anti-Jun, anti-Raf-1, monoclonal rat anti-mouse phospho-Raf-1 (series 338; Upstate Biotechnology), anti-MEK, and polyclonal rabbit anti-human phospho-MEK (Ser 217/221; Cell Signaling) antibodies.

Techniques: Incubation, Binding Assay, Western Blot, Immunoprecipitation

Journal:

Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

doi: 10.1128/MCB.22.22.7929-7941.2002

Figure Lengend Snippet: DEX inhibits Raf-1, MEK, and ERK-1/2 phosphorylation. Mouse thymocytes left untreated or pretreated for 6 h with DEX (100 nM) were stimulated for 1 h with plastic-bound anti-CD3 MAb. Western blotting was performed with an anti-pERK-1/2 (A), anti-pMEK-1/2 (B), anti-pRaf-1 (C), or anti-GILZ (D) antibody. Western blotting was also performed with an anti-ERK-1/2, anti-MEK-1/2, or anti-Raf-1 antibody to verify that no modulation of protein expression occurred or with β-tubulin to verify that equivalent amounts of proteins were loaded in all lanes.

Article Snippet: The primary antibodies were a polyclonal rabbit antiserum recognizing GILZ, a polyclonal rabbit anti-mouse phospho-ERK-1/2, and anti-mouse ERK-1/2 (Cell Signaling), anti-Fos, anti-Jun, anti-Raf-1, monoclonal rat anti-mouse phospho-Raf-1 (series 338; Upstate Biotechnology), anti-MEK, and polyclonal rabbit anti-human phospho-MEK (Ser 217/221; Cell Signaling) antibodies.

Techniques: Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: HDAC3-dependent Reversible Lysine Acetylation of Cardiac Myosin Heavy Chain Isoforms Modulates Their Enzymatic and Motor Activity *

doi: 10.1074/jbc.M110.163865

Figure Lengend Snippet: Myosin heavy chains are acetylated at lysine residues. A, myofibrillar proteins of mouse heart (25 μg) were incubated at 30 °C for 1 h in 1× HAT buffer containing 50 μm TSA, 50 mm NAM, 1 mm acetyl-CoA (AcCoA) where mentioned. The acetylation status of MHC was determined by Western blotting with the use of an anti-Ac-K antibody. Unless otherwise mentioned, all Western blots were stained with Coomassie Brilliant Blue to verify protein loading. B, porcine β-myosin (Sigma) (2 and 4 μg) was subjected to acetylation with PCAF in a HAT buffer containing TSA, NAM, and acetyl-CoA (as described in A). MHC acetylation and protein loading were examined by immunoblotting (IB) with the use of anti-Ac-K and anti-MHC antibodies. The lower panel shows loss of acetylation signal when Ac-K antibody was first incubated with acetyl-BSA. C, neonatal rat cardiomyocyte cultures were treated with 5 μm TSA, 50 mm NAM, and 10 μm acetyl-CoA (where mentioned) for 5 h. The myofibrillar fraction was prepared and analyzed for MHC acetylation by IB. The same blot was probed with anti-actin antibody for a loading control.

Article Snippet: The following antibodies and conjugates were used in this study: rabbit anti-acetyl lysine (9441, Cell Signaling/Millipore; 06-933, Upstate/Millipore); goat anti-actin (sc-1616, Santa Cruz Biotechnology), mouse anti-α-actinin (A7811, Sigma); rabbit anti-HDAC3 (ab2379 and ab16047, Abcam); HDAC3 blocking peptide (ab16279, Abcam), anti-histone 2A (2578, Cell Signaling), and anti-cardiac MHCs (ab15, Abcam).

Techniques: Incubation, Western Blot, Staining