For determining the expression of PLpro and TGF-β1 as well as nuclear translocalization of transcription factors, A549 cells transiently transfected with pSARS-PLpro or empty vector grew on the glass coverslip in 6-well at 37 °C. After 2 days incubation, transfected cells were fixed with 3.7% formaldehyde in phosphate buffered saline (PBS) for 1 h, blocked with 1% bovine serum albumin (BSA) in PBS for the other 1 h, and then incubated with specific primary antibodies at 4 °C overnight, including mouse anti-SARS PLpro, rabbit anti-TGF-β1 (Cell signaling), rabbit anti-NF-κB p65 (Abcam), rabbit anti-Sp-1, rabbit anti-Egr-1 (Cell Signaling), and rabbit anti-vimentin (GeneTex). Subsequently, cells were reacted with FITC-conjugated goat anti-mouse or rabbit IgG plus DAPI (4′,6-diamidino-2-phenylindole) in dark box for 2 h. After washing three times with PBS, photographs of stained cells were taken using the immunofluorescence microscopy (Olympus, BX50).
Rabbit anti egr 1
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-Egr-1 is a primary antibody that specifically binds to the Egr-1 (Early Growth Response 1) protein. Egr-1 is a transcription factor that plays a role in the regulation of gene expression in response to various stimuli, such as growth factors, cytokines, and cellular stress. This antibody can be used in applications like Western blotting, immunohistochemistry, and immunocytochemistry to detect and study the expression and localization of Egr-1 in biological samples.
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Lab products found in correlation
Rabbit anti tgf β1, by Cell Signaling Technology (2 mentions)
Rabbit anti vimentin, by GeneTex (2 mentions)
Rabbit anti sp1, by Cell Signaling Technology (1 mentions)
Rabbit anti nf κb p65, by Abcam (1 mentions)
Rabbit anti caspase 9, by Cell Signaling Technology (1 mentions)
Rabbit anti parp1, by Cell Signaling Technology (1 mentions)
Irdye800cw donkey anti mouse igg h l, by LI COR (1 mentions)
Rabbit anti c jun, by Cell Signaling Technology (1 mentions)
Rabbit anti smad2 3, by Cell Signaling Technology (1 mentions)
Mouse anti cytochrome c, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti c fos, by Cell Signaling Technology (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Irdye 800cw donkey anti rabbit igg h l, by LI COR (1 mentions)
Rabbit anti tubulin, by Cell Signaling Technology (1 mentions)
Rabbit anti p smad2 3, by Cell Signaling Technology (1 mentions)
Irdye 680lt donkey anti mouse igg h l, by LI COR (1 mentions)
Mouse anti snail, by Cell Signaling Technology (1 mentions)
Mouse anti caspase 8, by Cell Signaling Technology (1 mentions)
10 protocols using rabbit anti egr 1
1
Analyzing SARS-CoV-2 PLpro and TGF-β1 Expression
2
Electrophoretic Mobility Shift Assay for Rat DCC Gene Promoter
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Electrophoretic mobility shift assays (EMSAs) were performed as described before [21 (link), 22 (link)], by the Light-shift Chemiluminescent EMSA Kit (Pierce Chemical Co., Rockford, IL). A synthetic DNA double-stranded oligonucleotide probe (5′-biotin-CGGTACATGACACAGGCTGAC-5′) containing the sequence of the rat DCC gene promoter between nucleotides -101 and -91 bp (5′-CCAGCTCGCA-3′) was labeled with biotin and incubated with the nuclear extracts. Rabbit anti-Egr-1 (Cell Signaling technology Inc.) was used for supershift experiments. A chemiluminescent detection method with a luminal/enhancer solution and stable peroxide solution (Pierce Chemical Co., Rockford, IL) was used, as described by the manufacturer, and the membranes were exposed to X-ray films for 30 s to 5 min before development.
3
Western Blot Analysis of Signaling Proteins
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Cells were lysed in Laemmli sample buffer and run in 4–20% Mini-PROTEAN TGX precast protein gels (Bio-Rad Laboratories). The primary antibodies used were rabbit anti-Egr1 (no. 4154; Cell Signaling), rabbit anti–c-Fos (no. 2250; Cell Signaling), rabbit anti–c-Jun (no. 9165; Cell Signaling), mouse anti-Snail (no. 3895; Cell Signaling), rabbit anti-PARP1 (no. 9532; Cell Signaling), rabbit anti-Smad2/3 (no. 8685; Cell Signaling), rabbit anti-pSmad2/3 (no. 8828; Cell Signaling), mouse anti–cytochrome c (no. sc-13560; Santa Cruz Biotechnology), mouse anti-Cox4 (no. 11967; Cell Signaling), rabbit anti–caspase 9 (no. 9502; Cell Signaling), mouse anti–caspase 8 (no. 9746; Cell Signaling), rabbit anti-Tubulin (no. 2128; Cell Signaling), and mouse anti–α-Tubulin (T6199; Sigma). The secondary antibodies used were IRDye 800CW donkey anti–rabbit IgG (H+L), IRDye 680LT donkey anti–mouse IgG (H+L), and IRDye 800CW donkey anti–mouse IgG (H+L; LI-COR Biosciences). The blots were scanned on an Odyssey imaging system (LI-COR Biosciences). Cell treatment, sample collection, and Western blotting were repeated at least three times, and the representative blots are shown in the figures.
4
Immunohistochemical Analysis of Egr1 and P-CREB
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Mice were perfused transcardially with 50 ml PBS, followed by 50 ml of 4% paraformaldehyde and their brains were removed and post-fixed overnight in 4% PFA. Each brain was embedded in paraffin and 5 μm-thick coronal sections. Standard histological Hematoxylin-Eosin staining procedure was performed. Tissue sections were incubated overnight with rabbit anti-Egr1 (Cell Signaling Technology, Beverly, MA, USA) or rabbit anti—P-CREB (Ser133, 06-519, Millipore, Temecula, CA, USA) primary antibodies, and staining was revealed using biotinylated secondary antibodies and the ABC kit (Vector with diaminobenzidine (DAB; Sigma)). Individual cell numbers were quantified by Egr1, and P-CREB was quantified as the mean signal intensity using Image-Pro Plus 6.0 8 software (Media Cybernetics, Bethesda, MD, USA).
5
Western Blot Analysis of EGR1 Expression
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Cells were lysed in RIPA buffer (Sigma-Aldrich) supplemented with 1X Protease/Phosphatase Inhibitor Cocktail (Cell Signaling, Danvers, MA, United States) and separated on 4–20% gradient SDS/PAGE (Novex). Proteins were transferred on nitrocellulose membranes and incubated with primary antibodies overnight at 4°C. The primary antibodies used for Western blotting were rabbit anti-EGR1 (#4153S; Cell Signaling), rabbit anti-GAPDH (#5174S; Cell Signaling) and rabbit anti-β-actin (#4970S; Cell Signaling). The secondary antibody used was HRP linked anti-rabbit IgG (#7074P2; Cell Signaling). Chemiluminescence (ECL) was used to detect protein abundance.
6
Evaluating Inflammatory Mediators in Arthritis
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The concentration of IL-1β and TNF-α in articular cavity fluid was determined by the ELISA kit (R&D Systems) according to the manufacturer’s instructions. And tissues were washed three times with PBS, pulverized in liquid nitrogen, and lysed [20 (link)]. The homogenates (20 μg of protein) were separated by 8% SDS-PAGE and blotted on polyvinylidene fluori (PVDF) membranes (Bio–Rad Laboratories). Transblots were probed with the rabbit anti-Egr-1 (Cell Signaling technology Inc., 1:1000), rabbit anti-VEGF antibody (Santa Cruz Biotechnology, 1:400), rabbit anti-DCC antibody (Santa Cruz Biotechnology, 1:400), or rabbit anti-Netrin-1 antibody (Santa Cruz Biotechnology, 1:400) to examine the protein expression in the lysates. The amount of protein transferred onto the membranes was verified by immunoblotting for GAPDH (Santa Cruz Biotechnology, 1:500).
7
Protein Expression and Phosphorylation Analysis
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To determine protein expression and phosphorylation status, transfected A549 cells with empty vector pcDNA3.1 or pSARS-PLpro were harvested 2 days after transfection. Western blotting of cell lysates was accomplished, as described in our prior reports12 (link)13 (link). Resulting blots were probed with primary antibodies, including mouse polyclonal anti-E. coli synthesized PLpro, rabbit anti-vimentin (GeneTex), rabbit anti-TGF-β1 (Cell signaling), anti-α-SMA (Santa Cruz Biotechnology), rabbit anti-Egr-1, anti-phospho Erk1/2 (Thr202/Tyr204), anti-phospho p38 MAPK (Thr180/Tyr182), anti-phospho STAT3 (Ser727) (Cell Signaling), and anti-β-actin mAb (Abcam). Immune complexes were detected using HRP-conjugated goat anti-mouse or anti-rabbit IgG antibodies, as well as enhanced chemiluminescent HRP substrate (Millipore).
8
Molecular Mechanisms of Oxidative Stress
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SiRNAs were purchased from Shanghai Genepharma Co., Ltd. (China). Lipofectamine 2000 was purchased from Invitrogen (United States), Opti-MEM media was purchased from Life Technologies (United States). 2′,7′-Dichlorofluorescein acetyl acetate (DCFH-DA) was purchased from Sigma–Aldrich (United States). F2 was synthesized in our laboratory and dissolved in DMSO (≤0.1%). The following primary antibodies were purchased from Cell Signaling Technology (United States): rabbit anti-Egr-1, mouse anti-SIRT1, and rabbit anti-FOXO1 antibody. Rabbit anti-Ac-FOXO1 antibody was purchased from Santa Cruz Biotechnology (United States). Mouse β-actin antibody, anti-rabbit secondary antibodies and anti-mouse secondary antibodies were purchased from Wuhan Boster Biotechnology Limited Company (China). Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 594 goat anti-rabbit IgG were purchased from Life Technologies (United States). All reagent kits for real-time RT-PCR were purchased from TaKaRa Biotechnology (China). JC-1 was purchased from Beyotime Biotechnology (China).
9
Multimodal Imaging of Cellular Markers
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Samples were fixed by incubating with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature. After thorough washing with PBS, the fixed cells were permeabilized and blocked with Triton X-100 (0.3 %) and bovine serum albumin (3%) in PBS for 35 min at room temperature (RT). Then, the samples were incubated at 4°C for 48 hours with the following primary antibodies: mouse anti-tubulin β3 (1:1000; BioLegend), rabbit anti-GFAP (1:1000; Abcam), rabbit anti-Egr1 (1:1000; Cell Signaling Technology), and rabbit anti–β-catenin (Cell Signaling Technology). After washing with PBS, the resulting samples were stained with goat anti-rabbit immunoglobulin (IgG) [heavy and light chains (H + L)] secondary antibody or Alexa Fluor 546 (Invitrogen), goat anti-mouse IgG (H + L) secondary antibody, and Alexa Fluor 633 conjugate (Invitrogen) for 40 min at RT. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), and F-actin was labeled with Alexa Fluor 546 phalloidin (Thermo Fisher Scientific) if necessary. All fluorescence images were taken using Prairie Technologies 2-photon and confocal microsope, QuantEM 512SC camera, 60× and 40× objective lenses, and native Prairie View software and visualized by z-stack mode. All the image analysis and processing were performed with ImageJ and Imaris.
10
Autophagy Regulation by EGR1 Signaling
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The following antibodies were used: mouse anti-MAP1LC3B (Nanotools, 0231–100/LC3-5F10), rabbit anti-ATG7 (Cell Signaling Technology, 2631S), rabbit anti-RB1CC1/FIP200 (ITK diagnostics, A301-536A), rabbit anti-EGR1 (Cell Signaling Technology, 4154S), mouse anti-RPB1 (Euromedex; PB-7C2), rabbit anti-histone H3 acetyl K27 (Abcam, ab4729), rabbit anti-histone H3 acetyl K56 (Active Motif, 39,281), rabbit anti-histone H3 trimethyl K4 (Active Motif, 39,159), mouse anti-histone H3 (Active Motif, 39,763), mouse anti-TUBA4A/tubulin (Sigma-Aldrich, T9026). pMXs-hs-EGR1 was a gift from Shinya Yamanaka (Addgene, 52,724) [52 ]. For EGR1 knockdown, human SMARTpool EGR1 siRNA (Dharmacon, M-006526–01-0005) was used. pBABE-puro-mCherry-EFGP-LC3B was a gift from Jayanta Debnath (Addgene, 22,418) [53 ]. To increase the intensity of the fluorescence, the EEF1A1/EF1α promoter was cloned into the construct using NaeI restriction sites. bafilomycin A1 was obtained from Sigma-Aldrich (B1793). Hydroxychloroquine (HCQ) was obtained from Acros Organics (263,010,250).
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