Journal: The Journal of Biological Chemistry

Article Title: Microhomology-based CRISPR tagging tools for protein tracking, purification, and depletion

doi: 10.1074/jbc.RA119.008422

Figure Lengend Snippet: N-terminal tagging of the proteasome subunit RPN1 with HBTH and mCherry. A, HBTH-tagged RPN1 was detected using HRP-conjugated streptavidin and RPN1 antibodies. B, cells expressing HBTH-tagged RPN1 were lysed for proteasome activity assay using fluorogenic peptide as substrates. Fluorescent signals were normalized to total protein loaded to the assay. C, affinity purification of the proteasome using cells expressing HBTH-RPN1 and streptavidin beads. 19S and 20S proteasome subunits are visualized on a PVDF membrane with Amido Black stain. Mass spectrometry analysis confirmed that the pulldown had all proteasome subunits (Table S2). D, mCherry-RPN1 expression 3 days after puromycin selection.

Article Snippet: Proteasome activity assay The proteasome substrates SUC-LLVY-AMC, SUC-LLE-AMC, and SUC-ARR-AMC were purchased from Boston Biochem.

Techniques: Expressing, Activity Assay, Affinity Purification, Membrane, Staining, Mass Spectrometry, Selection

Journal: Nutrients

Article Title: A Bioassay-Guided Fractionation of Rosemary Leaf Extract Identifies Carnosol as a Major Hypertrophy Inducer in Human Skeletal Muscle Cells

doi: 10.3390/nu13124190

Figure Lengend Snippet: Proteasome activity is inhibited by carnosol. The proteasome chymotrypsin-like activity in protein extracts from myotubes (M19) ( A ) incubated (CO) or not (CTRL) with carnosol (1 μg/mL) for 48 h or ( B ) transfected with a control siRNA (siCTRL) or a siRNA against MuRF1 (siMuRF1) was measured by monitoring the hydrolysis of Suc-LLVY-AMC peptide through quantification of AMC fluorescence in the presence or not of bortezomib, a specific proteasome inhibitor; * p < 0.05. Data are the mean ± SD of four replicates. M19, 19-year-old male donor.

Article Snippet: Measurement of proteasome activity: 10 μg of myotube extracts/well for each condition were deposited in 96-well plates and incubated with proteasome substrate alone (Suc-LLVY-AMC- Bachem AG ref-I-1395.0025) or proteasome substrate and the 20S proteasome inhibitor bortezomib (MedChemExp ref: HY-10227/CS-1039) at 20 μM final.

Techniques: Activity Assay, Incubation, Transfection, Control, Fluorescence

Journal: Autophagy

Article Title: Azithromycin attenuates myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4

doi: 10.1080/15548627.2017.1328348

Figure Lengend Snippet: AZM induces proteasomal degradation of NOX4. (A) WB using anti-NOX4 and anti-ACTB of cell lysates from control (lane 1, 3) and AZM- (10 μg/ml) treated (lane 2, 4) LF in the presence (lane 1, 2) or absence of MG132 (lane 3, 4). Protein samples were collected after 3 h treatment with AZM and MG132. Shown in the lower panel is the average ( ± SEM) taken from 6 independent experiments shown as relative expressions. *p < 0.05. (B) WB of cell lysates from TGFB1- (2 ng/ml) treated (lane 1, 2, 3, 4) and AZM (10 μg/ml) treated (lane 2, 4) LF in the presence (lane 1, 2) or absence of MG132 (lane 3, 4). MG132 and AZM treatment was started 1 h before TGFB1 stimulation. Shown in the lower panel is the average ( ± SEM) taken from 5 independent experiments shown as relative expressions. *p < 0.05. (C) Changes of 20S proteasome activity in response to AZM (10 μg/ml) treatment. After 24-h treatment with MG132 and AZM, cell lysates for measuring 20S proteasome activity were collected. Line plots show the measured relative value of fluorescence units (RFU) of average ( ± SD). The fluorescence level in the control treated cells was designated as 1.0 and shown in the lower panel is the average ( ± SEM) taken from 6 independent experiments. *p < 0.05. (D) WB using anti-K48 ubiquitin and anti-ACTB of cell lysates from control (lane 1), TGFB1- (2 ng/ml) treated (lane 2), and AZM- (10 μg/ml) treated (lane 3) LF. Shown in the lower panel is the average ( ± SEM) taken from 8 independent experiments shown as relative expressions. *p < 0.05. (E) Colocalization analysis of confocal laser scanning microscopy images of NOX4 and ubiquitin staining in LF. LF were treated with AZM (10 μg/ml) and TGFB1 (2 ng/ml) for 4 h in the presence of MG132. Staining was performed using an anti-NOX4 antibody and anti-ubiquitin antibody. Bar: 10 µm. (F) WB using anti-STUB1 and anti-ACTB of cell lysates from TGFB1- (2 ng/ml) treated (lane 3, 4) and AZM- (10 μg/ml) treated (lane 2, 4) LF. Shown in the lower panel is the average ( ± SEM) taken from 5 independent experiments shown as relative expressions. *p < 0.05. (G) Colocalization analysis of confocal laser scanning microscopy images of NOX4 and ubiquitin staining in LF. LF were transfected with control siRNA and STUB1 siRNA, and treatment with AZM (10 μg/ml) and TGFB1 (2 ng/ml) was started 48 h post-siRNA transfection. Bar: 10 µm. (H) WB using cell lysates from control and STUB1 siRNA-transfected LF. TGFB1 (2 ng/ml) and AZM (10 μg/ml) treatment were started 48 h post-siRNA transfection and protein samples were collected after 24 h treatment. Shown in the lower panel is the average ( ± SEM) taken from 7 independent experiments shown as relative expressions. *p < 0.05.

Article Snippet: The 20S proteasome was isolated from LF and lung tissue lysate by using hHR23B ubiquitin-like domain resin (UbL-resin; Cyclex Co., Ltd. CY-7002), and 20S proteasome activity was measured by adding a fluorogenic proteasome substrate (Suc-LLVY-AMC; Cyclex Co., Ltd. CY-7002).

Techniques: Activity Assay, Fluorescence, Confocal Laser Scanning Microscopy, Staining, Transfection

Journal: Autophagy

Article Title: Azithromycin attenuates myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4

doi: 10.1080/15548627.2017.1328348

Figure Lengend Snippet: Effect of AZM on TGFB-induced autophagy and TGFB-mediated UPR and proteasome activation in LF. (A) WB using anti-LC3 and anti-ACTB of cell lysates from control (lane 1, 2), TGFB1-treated (lane 3, 4, 7, 8) and AZM-treated (lane 5, 6, 7, 8) LF in the presence or absence of protease inhibitors (E64d, pepstatin A). Protein samples were collected after 24 h treatment with AZM (10 μg/ml) and TGFB1 (2 ng/ml). In the lower panel is the average ( ± SEM) taken from 3 independent experiments shown as relative expression. *p < 0.05. (B) WB using anti-SQSTM1 and anti-ACTB of cell lysates from control (lane 1), TGFB1- (2 ng/ml) treated (lane 2, 4), and AZM (10 μg/ml) treated (lane 3, 4) LF. Protein samples were collected after 24 h treatment. In the lower panel is the average ( ± SEM) taken from 3 independent experiments shown as relative expression. *p < 0.05. (C) WB using anti-HSPA5, anti-p-ERN1, anti-ATF4, anti-ATF6, and anti-ACTB of cell lysates from control (lane 1), TGFB1- (2 ng/ml) treated (lane 2, 4), and AZM (10 μg/ml) treated (lane 3, 4) LF. Protein samples were collected after 24 h treatment. In the right panels are the average ( ± SEM) taken from 3 independent experiments shown as relative expression. *p < 0.05. (D) Changes of 20S proteasome activity by TGFB1 and AZM treatment. Cell lysates for measuring 20S proteasome activity were collected after 24 h treatment with control, TGFB1 (2 ng/ml), and concomitant TGFB1 and AZM (10 μg/ml). Shown in the right panel is the average ( ± SEM) taken from 3 independent experiments. *p < 0.05.

Article Snippet: The 20S proteasome was isolated from LF and lung tissue lysate by using hHR23B ubiquitin-like domain resin (UbL-resin; Cyclex Co., Ltd. CY-7002), and 20S proteasome activity was measured by adding a fluorogenic proteasome substrate (Suc-LLVY-AMC; Cyclex Co., Ltd. CY-7002).

Techniques: Activation Assay, Expressing, Activity Assay

Journal: Autophagy

Article Title: Azithromycin attenuates myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4

doi: 10.1080/15548627.2017.1328348

Figure Lengend Snippet: Autophagy inhibition and enhanced unfolded protein responses (UPR) by AZM in association with STUB1 regulation and proteasome activation in LF. (A) WB using anti-LC3B and anti-ACTB of cell lysates from control (lane 1, 2) and AZM (lane 3, 4) treated LF in the presence or absence of protease inhibitors (E64d, pepstatin A). Protein samples were collected after 24-h treatment with AZM (10 μg/ml). In the lower panel is the average ( ± SEM) taken from 3 independent experiments shown as relative expression. *p < 0.05. (B) Colocalization analysis of mRFP and EGFP by using LF expressing mRFP-GFP tandem fluorescent-tagged LC3B. After 24 h treatment with bafilomycin A1 (Baf A1; 10 nM) and AZM (10 μg/ml), fixed LF were evaluated by confocal laser scanning microscopy. Bar: 20 μm. (C) WB using anti-SQSTM1 and anti-ACTB of cell lysates from control (lane 1) and AZM (10 μg/ml) treated (lane 2) LF. Protein samples were collected after 24 h treatment. In the lower panel is the average ( ± SEM) taken from 3 independent experiments shown as relative expression. *p < 0.05. (D) WB using anti-ATG5, anti-HSPA5, anti-p-ERN1, anti-ATF4, anti-ATF6, anti-STUB1, and anti-ACTB of cell lysates from control siRNA (lane 1) and ATG5 siRNA- (lane 2) transfected LF. Protein samples were collected after 72 h transfection. In the right panels are the average ( ± SEM) taken from 4 independent experiments shown as relative expression. *p < 0.05. (E) Changes of 20S proteasome activity by ATG5 knockdown. Cell lysates for measuring 20S proteasome activity were collected after 72-h transfection with control and ATG5 siRNA. Line plots show the measured relative value of fluorescence units (RFU) of the averages ( ± SD). Shown in the lower panel is the average ( ± SEM) taken from 3 independent experiments. *p < 0.05. (F) WB of cell lysates from control (lane 1) and AZM (10 μg/ml) treated (lane 2) LF. In the right panels are the average ( ± SEM) taken from 3 independent experiments shown as relative expression. *p < 0.05. (G) WB of cell lysates from control (lane 1) and tunicamycin (TM; 0.1 μg/ml) treated LF (lane 2). Protein samples were collected after 24 h treatment with TM. In the right panels are the average ( ± SEM) taken from 4 independent experiments shown as relative expression. *p < 0.05. (H) Changes of 20S proteasome activity by TM treatment. Cell lysates for measuring 20S proteasome activity were collected after 24 h treatment with control and TM. Line plots show the measured relative value of fluorescence units (RFU) of the averages ( ± SD). Shown in the right panel is the average ( ± SEM) taken from 3 independent experiments. *p < 0.05. (I) 20S proteasome activity of concomitant AZM and TM treatment, and concomitant ATG5 knockdown and TM treatment. AZM and TM treatments were started 48 h post-siRNA transfection and cell lysates for measuring 20S proteasome activity were collected after 24 h treatment with AZM and TM. Shown in the panel is the average ( ± SEM) taken from 3 independent experiments. *p < 0.05.

Article Snippet: The 20S proteasome was isolated from LF and lung tissue lysate by using hHR23B ubiquitin-like domain resin (UbL-resin; Cyclex Co., Ltd. CY-7002), and 20S proteasome activity was measured by adding a fluorogenic proteasome substrate (Suc-LLVY-AMC; Cyclex Co., Ltd. CY-7002).

Techniques: Inhibition, Activation Assay, Expressing, Confocal Laser Scanning Microscopy, Transfection, Activity Assay, Fluorescence

Journal: Autophagy

Article Title: Azithromycin attenuates myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4

doi: 10.1080/15548627.2017.1328348

Figure Lengend Snippet: Effect of AZM on bleomycin-induced lung fibrosis development in mice. (A) Body weight (BW) changes after BLM treatment. BW at d 0 before treatment was designated as 1.0. (B) Shown in the panel is the average ( ± SEM) soluble collagen measurement from Sircol assay using control (n = 12), BLM-treated (n = 15), and BLM-treated with subsequent AZM injection mouse lungs (n = 15) at d 21. Open bar is control, filled bar is BLM-treated, and horizontal crosshatched bar is BLM-treated with subsequent AZM injection. *p < 0.05. (C) Photomicrographs of Masson trichrome staining of mouse lungs at d 21. Upper panels are low magnification view of original magnification × 40. Lower panels are high magnification view of original magnification × 100. The right panel shows the average ( ± SEM) percentage of positively stained area quantified using ImageJ. *p < 0.05. (D) Immunohistochemical staining of NOX4 in mouse lungs at day 21. Original magnification × 100. (E) WB using anti-NOX4 and anti-ACTB of lung homogenates. Lower panel is the average ( ± SEM) shown as relative expressions. Treatment groups were composed of control (n = 7), BLM-treated (n = 6), and BLM-treated with subsequent AZM injection mouse lungs (n = 6). *p < 0.05. (F) WB using anti-K48 ubiquitin, anti-STUB1, and anti-ACTB of lung homogenates. Lower panels are the average ( ± SEM) shown as relative expressions. Treatment groups were composed of control (n = 4), BLM-treated (n = 7), and BLM-treated with subsequent AZM injection mouse lungs (n = 8). *p < 0.05. (G) 20S proteasome activity of mouse lungs. Mouse lungs for measuring 20S proteasome activity were collected at d 21. The fluorescence level in the control-treated lung homogenate from one representative mouse was designated as 1.0. Shown panel is the average ( ± SEM) and treatment groups were composed of the same number of mice (n = 5). *p < 0.05.

Article Snippet: The 20S proteasome was isolated from LF and lung tissue lysate by using hHR23B ubiquitin-like domain resin (UbL-resin; Cyclex Co., Ltd. CY-7002), and 20S proteasome activity was measured by adding a fluorogenic proteasome substrate (Suc-LLVY-AMC; Cyclex Co., Ltd. CY-7002).

Techniques: Injection, Staining, Immunohistochemical staining, Activity Assay, Fluorescence

Journal: Autophagy

Article Title: Azithromycin attenuates myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4

doi: 10.1080/15548627.2017.1328348

Figure Lengend Snippet: Hypothetical model of Azithromycin-mediated inhibition of myofibroblast differentiation and lung fibrosis development. AZM-mediated NOX4 degradation can result from enhanced proteasome activation and NOX4 ubiquitination by STUB1, an E3 ubiquitin ligase. Autophagy inhibition by AZM is involved in enhanced ubiquitination of NOX4 via increased protein levels of STUB1. The increase in proteasome activity appeared to be mainly attributable to an increased UPR caused by AZM, which may be resulting from functional crosstalk between proteostasis machineries. AZM can be a novel modality of IPF treatment of targeting NOX4 degradation resulting from modulation of crosstalk between proteostasis machineries.

Article Snippet: The 20S proteasome was isolated from LF and lung tissue lysate by using hHR23B ubiquitin-like domain resin (UbL-resin; Cyclex Co., Ltd. CY-7002), and 20S proteasome activity was measured by adding a fluorogenic proteasome substrate (Suc-LLVY-AMC; Cyclex Co., Ltd. CY-7002).

Techniques: Inhibition, Activation Assay, Activity Assay, Functional Assay

Journal:

Article Title: Evidence Against a Direct Antiviral Activity of the Proteasome during the Early Steps of HIV-1 Replication

doi: 10.1016/j.virol.2007.01.018

Figure Lengend Snippet: Left panel: P4.R5 cells were pretreated with MG-132 (25 μM) or nothing for 5 hours. The cells were then washed and exposed to 150 μM Suc-LLVY-AMC (with or without 25 μM MG-132) immediately or after a 2- or 5-hour incubation. Right panel: P4.R5 cells were pretreated with bortezomib (1 μM) or nothing for 5 hours. The cells were then washed and exposed to 150 μM Suc-LLVY-AMC (with or without 1 μM bortezomib) immediately or after a 2- or 5-hour incubation. Measurements were taken every 3 minutes in a fluorometer; the cells were maintained at 37° C. Each point represents the average of 3 measurements (C.V. < 1% in all cases). “LLVY” is suc-LLVY-AMC.

Article Snippet: Fluorogenic Proteasome Substrate III Suc-Leu-Leu-Val-Tyr-AMC (Suc-LLVY-AMC) was purchased from Axxora (#BST-S-280) and resuspended in DMSO.

Techniques: Incubation