Journal: Cancer Communications

Article Title: Cytoplasmic YAP1‐mediated ESCRT‐III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

doi: 10.1002/cac2.12417

Figure Lengend Snippet: EGCG promoted retention of YAP1 in the cytoplasm by activating the Hippo pathway and promoting autophagy in breast cancer cells. (A) Natural small‐molecule compounds predicted to activate the Hippo pathway in breast cancer cell lines. (B) Bioinformatics analysis of pathways related to EGCG. (C) WB analysis was performed to examine expression of Hippo pathway components and YAP1 target genes ( CTGF and CYR61 ) in MCF7 and MDA‐MB‐231 cells treated with EGCG of various concentrations for 6 h. (D) Expression levels of YAP1 and p‐YAP1 were determined in the nucleus and cytoplasm of MCF7 and MDA‐MB‐231 cells via WB analysis. LaminB1 and β‐actin were used as extraction controls for the nucleus and cytoplasm, respectively. (E‐F) Expression levels of the autophagy markers LC3 and p62 were detected by WB in MCF7 and MDA‐MB‐231 cells after treatment with EGCG of various concentrations or for different lengths of time. (G) IF staining images showing LC3 fluorescence puncta and quantitative analysis of MCF7 and MDA‐MB‐231 cells treated with EGCG (50 µg/mL, 6 h). DAPI labelled with blue fluorescent signal was used to mark the nucleus, while green fluorescent signal was used to label LC3. (H) Autophagic structures (indicated with red arrows) were detected with TEM in MCF7 and MDA‐MB‐231 cells treated with EGCG (50 µg/ml, 6 h). Data are from three independent experiments and are shown as the mean ± standard deviation. ** P < 0.01 (Student's t ‐test). Abbreviations: YAP1, Yes1‐associated transcriptional regulator; EGCG, epigallocatechin gallate; WB, Western blotting; MST1, macrophage stimulating 1; p‐MST1, phosphorylated‐macrophage stimulating 1; MOB1A: MOB kinase activator 1A; p‐MOB1A: phosphorylated‐MOB1A; CTGF, connective tissue growth factor; CYR61, cysteine rich angiogenic inducer 61; LC3, microtubule‐associated protein 1 light chain 3; SQSTM1/p62, sequestosome 1; IF, immunofluorescence staining; DAPI, 4',6‐diamidino‐2‐phenylindole; TEM, transmission electron microscopy.

Article Snippet: Antibodies for YAP1 (13584‐1‐AP, 66900‐1‐Ig; 1:1000 for WB, 1:200 for immunohistochemistry [IHC], 1:100 for IF), NEDD4 like E3 ubiquitin protein ligase (NEDD4L, 13690‐1‐AP, 1:1000 for WB, 1:400 for IHC), sequestosome 1 (SQSTM1/p62, 18420‐1‐AP, 1:1000 for WB, 1:50 for IHC) and CYR61 (26689‐1‐AP, 1:1000 for WB, 1:50 for IHC) were purchased from Proteintech Group (Rosemont, IL, USA).

Techniques: Expressing, Extraction, Staining, Fluorescence, Standard Deviation, Western Blot, Immunofluorescence, Transmission Assay, Electron Microscopy

Journal: Cancer Communications

Article Title: Cytoplasmic YAP1‐mediated ESCRT‐III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

doi: 10.1002/cac2.12417

Figure Lengend Snippet: Cytoplasmic YAP1 promoted autophagic cell death in vivo. (A) Tumor‐bearing nude mice from different treatment groups ( n = 6 per group). (B) Tumors taken from mice in different treatment groups. (C) The weight of tumors from mice in each treatment group. (D) The tumor volume from mice in each treatment group over time. (E) WB analysis was performed to detect the expression of YAP1, p‐YAP1, the autophagy markers LC3 and p62, and YAP1 target genes CTGF and CYR61 in tumor tissues. (F) IHC staining of LC3, p62, YAP1, CTGF, and CYR61 in tumor tissues from mice in different treatment groups. (G) Autophagic structures (indicated with red arrows) in tumor tissues from the control and EGCG treatment groups were detected with TEM. Data are shown as the mean ± standard deviation. *** P < 0.001 (Student's t ‐test and one‐way ANOVA). Abbreviations: EGCG, epigallocatechin gallate; LC3, microtubule‐associated protein 1 light chain 3; SQSTM1/p62, sequestosome 1; WB, Western blotting; YAP1, Yes1‐associated transcriptional regulator; CTGF, connective tissue growth factor; CYR61, cysteine rich angiogenic inducer 61; IHC, immunohistochemistry; TEM, transmission electron microscopy; ANOVA, analysis of variance.

Article Snippet: Antibodies for YAP1 (13584‐1‐AP, 66900‐1‐Ig; 1:1000 for WB, 1:200 for immunohistochemistry [IHC], 1:100 for IF), NEDD4 like E3 ubiquitin protein ligase (NEDD4L, 13690‐1‐AP, 1:1000 for WB, 1:400 for IHC), sequestosome 1 (SQSTM1/p62, 18420‐1‐AP, 1:1000 for WB, 1:50 for IHC) and CYR61 (26689‐1‐AP, 1:1000 for WB, 1:50 for IHC) were purchased from Proteintech Group (Rosemont, IL, USA).

Techniques: In Vivo, Expressing, Immunohistochemistry, Control, Standard Deviation, Western Blot, Transmission Assay, Electron Microscopy

Journal: Frontiers in immunology

Article Title: Spermidine attenuates chondrocyte inflammation and cellular pyroptosis through the AhR/NF-κB axis and the NLRP3/caspase-1/GSDMD pathway.

doi: 10.3389/fimmu.2024.1462777

Figure Lengend Snippet: FIGURE 4 Effects of SPD on NF-kB signaling pathway. (A-C) Western blot and semi-quantitative analysis of p-IkBa, IkBa, p-p65, and p65. (D) Immunofluorescence to observe NF-kB P65 nuclear translocation. We stained the nuclei using DAPI (blue) and labeled NF-kB p65 using anti-NF-kB p65 rabbit fluorescent antibody (green), while labeling the cytoskeleton using YF®594-Phalloidin. Significant NF-kB p65 nuclear translocation was observed in the IL-1b group. pretreatment with SPD reversed IL-1b-induced nuclear translocation, whereas no significant changes were observed when SPD was applied alone. *p < 0.05, **p < 0.01 vs. the control group; #p < 0.05, ##p < 0.01 vs. the IL-1b group.

Article Snippet: After three washes with trisbuffered saline (TBS) containing 0.1% Tween-20 (TBST) for 10 min, overnight incubation was carried out with primary antibodies targeting Phospho-NF-kB p65 (1:1000; CST, #3033), NF-kB p65 (1:1000; CST, #8242), Phospho-IkBa (1:1000; CST, #2859), IkBa (1:1000; CST, #9242), iNOS (1:1000; Abcam, ab178945), COX2 (1:1000; Abcam, ab179800), Collagen II (1:1000; Abcam, ab34712), MMP13 (1:2000; Proteintech, 18165-1-AP), MMP3 (1:1000; Proteintech, 17873-1-AP), AhR (1:1000; Proteintech, 67785-1), ADAMTS-5 (1:1000; Abcam, ab41037), NLRP3 (1:1000; Proteintech, 19771-1-AP), Caspase-1/p20/ p10 (1:4000; Proteintech, 22915-1-AP), GSDMD (1:1000; #AF4012, Affinity Biosciences), and GAPDH (1:20000; Proteintech, 10494-1- AP).

Techniques: Western Blot, Translocation Assay, Staining, Labeling, Control

Journal: Frontiers in immunology

Article Title: Spermidine attenuates chondrocyte inflammation and cellular pyroptosis through the AhR/NF-κB axis and the NLRP3/caspase-1/GSDMD pathway.

doi: 10.3389/fimmu.2024.1462777

Figure Lengend Snippet: FIGURE 5 The protective effect of the SPD depends on the AhR. (A-C) Western blot and qRT-PCR analysis for AhR knockdown. (D-G) Expression of IL-6, TNF- a and PGE2 in cell suspensions by ELISA and NO by Griess reaction. (H-P) Western blot and semi-quantitative analysis of COX-2, iNOS, MMP-3, MMP-13, Collagen-II, ADAMTS-5 and p-IkBa, IkBa, p-p65 and p65. *p < 0.05, **p < 0.01 vs. the control group; #p < 0.05, ##p < 0.01 vs. the IL-1b group; ※p < 0.05, ※※p < 0.01 vs. the IL-1b+SPD group.

Article Snippet: After three washes with trisbuffered saline (TBS) containing 0.1% Tween-20 (TBST) for 10 min, overnight incubation was carried out with primary antibodies targeting Phospho-NF-kB p65 (1:1000; CST, #3033), NF-kB p65 (1:1000; CST, #8242), Phospho-IkBa (1:1000; CST, #2859), IkBa (1:1000; CST, #9242), iNOS (1:1000; Abcam, ab178945), COX2 (1:1000; Abcam, ab179800), Collagen II (1:1000; Abcam, ab34712), MMP13 (1:2000; Proteintech, 18165-1-AP), MMP3 (1:1000; Proteintech, 17873-1-AP), AhR (1:1000; Proteintech, 67785-1), ADAMTS-5 (1:1000; Abcam, ab41037), NLRP3 (1:1000; Proteintech, 19771-1-AP), Caspase-1/p20/ p10 (1:4000; Proteintech, 22915-1-AP), GSDMD (1:1000; #AF4012, Affinity Biosciences), and GAPDH (1:20000; Proteintech, 10494-1- AP).

Techniques: Western Blot, Quantitative RT-PCR, Knockdown, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in immunology

Article Title: Spermidine attenuates chondrocyte inflammation and cellular pyroptosis through the AhR/NF-κB axis and the NLRP3/caspase-1/GSDMD pathway.

doi: 10.3389/fimmu.2024.1462777

Figure Lengend Snippet: FIGURE 6 SPD ameliorates IL-1b-induced chondrocyte pyroptosis by inhibiting the NLRP3/caspase-1/GSDMD pathway. (A-E) Western blot measuring the differences in the expression levels of cellular pyroptosis-related proteins in each group. To elucidate whether SPD ameliorates chondrocyte pyroptosis by inhibiting AhR/NF-kB, NF-kB p65 overexpression plasmid was used to characterize the effect of the AhR/NF-kB pathway. (F-J) The inflammation-associated cellular pyroptosis proteins NLRP3, caspase-1, and GSDMD were significantly increased in the IL-1b group compared with the CG group, whereas downregulation of cellular pyroptosis was observed in the SPD group. Administration of P65 overexpression plasmid (OE group) blocked the therapeutic effect of SPD. (K-L) Flow cytometry showing chondrocytes labeled with caspase-1 fluorescence inhibitor probe FLICA 660-YVAD-FMK. IL-1b induces chondrocyte pyroptosis. SPD significantly alleviated IL-1b-induced chondrocyte death. (M) Transmission electron microscopy (TEM) images of chondrocytes from different treatment groups, such as cytoplasmic edema, swelling and rupture of cell membrane, nuclear consolidation, and organelle cavitation were more obvious in the IL-1b group; cell morphology of the SPD pretreatment group was significantly improved. Black arrows indicate cell membrane swelling; white arrows indicate organelle cavitation (Low magnification: ×2.0k, high magnification: ×10.0k). *p < 0.05, **p < 0.01 vs. the control group; #p < 0.05, ##p < 0.01 vs. the IL-1b group; ※p < 0.05, ※※p < 0.01 vs. the IL- 1b+SPD group.

Article Snippet: After three washes with trisbuffered saline (TBS) containing 0.1% Tween-20 (TBST) for 10 min, overnight incubation was carried out with primary antibodies targeting Phospho-NF-kB p65 (1:1000; CST, #3033), NF-kB p65 (1:1000; CST, #8242), Phospho-IkBa (1:1000; CST, #2859), IkBa (1:1000; CST, #9242), iNOS (1:1000; Abcam, ab178945), COX2 (1:1000; Abcam, ab179800), Collagen II (1:1000; Abcam, ab34712), MMP13 (1:2000; Proteintech, 18165-1-AP), MMP3 (1:1000; Proteintech, 17873-1-AP), AhR (1:1000; Proteintech, 67785-1), ADAMTS-5 (1:1000; Abcam, ab41037), NLRP3 (1:1000; Proteintech, 19771-1-AP), Caspase-1/p20/ p10 (1:4000; Proteintech, 22915-1-AP), GSDMD (1:1000; #AF4012, Affinity Biosciences), and GAPDH (1:20000; Proteintech, 10494-1- AP).

Techniques: Western Blot, Expressing, Over Expression, Plasmid Preparation, Flow Cytometry, Labeling, Transmission Assay, Electron Microscopy, Membrane, Control

Journal: Frontiers in immunology

Article Title: Spermidine attenuates chondrocyte inflammation and cellular pyroptosis through the AhR/NF-κB axis and the NLRP3/caspase-1/GSDMD pathway.

doi: 10.3389/fimmu.2024.1462777

Figure Lengend Snippet: FIGURE 7 SPD attenuates the progression of OA in a rat model. (A-C) A representative image of H&E, saffron O-solid green and toluidine blue staining of cartilage sections from different groups. (D) Articular cartilage Pritzker score in different groups of rats. (E) The modified Mankin score for different groups of cartilage. (F) The cartilage OARSI scores of different groups. (G-L) Immunohistochemical staining and percentage of positive cells for IL-6, NF-kB p65, Caspase-1, NLRP3, Gasdermin-D and Collagen II. Scale bar = 20 mm, ×400. *p < 0.05, **p < 0.01 vs. the control group; #p < 0.05, ##p < 0.01 vs. the ACLT group.

Article Snippet: After three washes with trisbuffered saline (TBS) containing 0.1% Tween-20 (TBST) for 10 min, overnight incubation was carried out with primary antibodies targeting Phospho-NF-kB p65 (1:1000; CST, #3033), NF-kB p65 (1:1000; CST, #8242), Phospho-IkBa (1:1000; CST, #2859), IkBa (1:1000; CST, #9242), iNOS (1:1000; Abcam, ab178945), COX2 (1:1000; Abcam, ab179800), Collagen II (1:1000; Abcam, ab34712), MMP13 (1:2000; Proteintech, 18165-1-AP), MMP3 (1:1000; Proteintech, 17873-1-AP), AhR (1:1000; Proteintech, 67785-1), ADAMTS-5 (1:1000; Abcam, ab41037), NLRP3 (1:1000; Proteintech, 19771-1-AP), Caspase-1/p20/ p10 (1:4000; Proteintech, 22915-1-AP), GSDMD (1:1000; #AF4012, Affinity Biosciences), and GAPDH (1:20000; Proteintech, 10494-1- AP).

Techniques: Staining, Immunohistochemical staining, Control

Journal: Frontiers in immunology

Article Title: Spermidine attenuates chondrocyte inflammation and cellular pyroptosis through the AhR/NF-κB axis and the NLRP3/caspase-1/GSDMD pathway.

doi: 10.3389/fimmu.2024.1462777

Figure Lengend Snippet: FIGURE 8 Mechanism of SPD inhibition of NF-kB signaling via AhR receptors and attenuation of chondrocyte inflammation and pyroptosis via NLRP3/caspase- 1/GSDMD signaling pathway. A rat OA model was constructed using ACLT. Changes in the levels of inflammatory factors in animal samples were observed by intraperitoneal injection of SPD. We found that the drug entered the cells and bound to AhR targeting to inhibit chondrocyte inflammation and inflammation-associated cellular pyroptosis, thereby ameliorating the pathologic changes in OA. In vitro, SPD inhibited chondrocyte inflammation and inflammation-associated cellular pyroptosis by blocking the activation of AhR/NF-kB and NLRP3/caspase-1/GSDMD signaling pathways in IL-1b-treated chondrocytes.

Article Snippet: After three washes with trisbuffered saline (TBS) containing 0.1% Tween-20 (TBST) for 10 min, overnight incubation was carried out with primary antibodies targeting Phospho-NF-kB p65 (1:1000; CST, #3033), NF-kB p65 (1:1000; CST, #8242), Phospho-IkBa (1:1000; CST, #2859), IkBa (1:1000; CST, #9242), iNOS (1:1000; Abcam, ab178945), COX2 (1:1000; Abcam, ab179800), Collagen II (1:1000; Abcam, ab34712), MMP13 (1:2000; Proteintech, 18165-1-AP), MMP3 (1:1000; Proteintech, 17873-1-AP), AhR (1:1000; Proteintech, 67785-1), ADAMTS-5 (1:1000; Abcam, ab41037), NLRP3 (1:1000; Proteintech, 19771-1-AP), Caspase-1/p20/ p10 (1:4000; Proteintech, 22915-1-AP), GSDMD (1:1000; #AF4012, Affinity Biosciences), and GAPDH (1:20000; Proteintech, 10494-1- AP).

Techniques: Inhibition, Construct, Injection, In Vitro, Blocking Assay, Activation Assay, Protein-Protein interactions

Journal: Cell

Article Title: The Golgi Outpost Protein TPPP Nucleates Microtubules and is Critical for Myelination

doi: 10.1016/j.cell.2019.08.025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Compound ThermoFisher Scientific Cat#23-730-571 VECTASHIELD Antifade Mounting Medium with DAPI Vector Laboratories Cat#H-1200 ProLong Gold Antifade Mountant ThermoFisher Scientific Cat# {"type":"entrez-protein","attrs":{"text":"P36934","term_id":"549428","term_text":"P36934"}} P36934 Precision Cover Glasses Thickness No. 1.5H (for super-resolution microscopy) Marienfeld Cat#0117520 35-mm Dish No. 1.5 Coverslip 14-mm Glass Diameter Poly-D-Lysine Coated MatTek Corporation Cat#P35GC-1.5–14-C 300 Mesh Copper Grid with Formvar/Carbon Coating/Film Electron Microscopy Sciences Cat#FCF300-Cu Goat Anti-Rabbit IgG 5-nm Gold Beads BBI Solutions Cat#EM.GAR5 OMIX C18 Pipette Tips Agilent Technologies Cat#A570033100 EasySpray C18 column (3-μm, 75 μm diameter × 150 mm length) ThermoFisher Scientific Cat#ES800A Open in a separate window KEY RESOURCES TABLE

Techniques: Western Blot, Staining, Recombinant, Affinity Chromatography, Fluorescence, Immunoprecipitation, Sequencing, Modification, Knock-Out, Plasmid Preparation, Software, Imaging, Microscopy, Electron Microscopy, Transferring

Journal: iScience

Article Title: PKR deficiency delays vascular aging via inhibiting GSDMD-mediated endothelial cell hyperactivation

doi: 10.1016/j.isci.2022.105909

Figure Lengend Snippet: Knockout of PKR inhibits the transforming phenotype of VSMC in aging vessel (A) Representative immunoblots and densitometric analysis of arterial α-SMA, SM22α, caldesmon, calponin, thrombospondin and osteopontin level in WT and PKR knockout mice of different age (n = 4). (B) Representative photomicrographs and statistical analysis of serial sections of arteries from WT and PKR knockout mice of different age (n = 5). Sections were immunohistochemical stained for the α-SMA, SM22α and osteopontin (n = 5). Scale Bar = 50 μm. ∗ p< 0.05; ∗∗ p< 0.01; ∗∗∗ p< 0.001 (two-way ANOVA test). Data are represented as mean ± SEM.

Article Snippet: The membranes were blocked with 5% fat-free milk before incubation with primary antibodies specifically recognizing PKR (ab32052, 1:1000, Abcam), p-PKR (ab32036, 1:1000, Abcam), human GSDMD (ab210070, 1:1000, Abcam), rabbit anti-human N-GSDMD antibody (EPR20829-408) (ab215203, 1:1000, Abcam) and IL-1β (MAB201, 1:1000, R&D system), α-SMA (1:1000, Proteintech), SM22α (1:1000, Proteintech), calponin (1:1000, Proteintech), caldesmon (1:1000, Proteintech), thrombospondin (1:1000, Proteintech) and osteopontin (1:1000, Proteintech).

Techniques: Knock-Out, Western Blot, Immunohistochemical staining, Staining

Journal: iScience

Article Title: PKR deficiency delays vascular aging via inhibiting GSDMD-mediated endothelial cell hyperactivation

doi: 10.1016/j.isci.2022.105909

Figure Lengend Snippet: Silencing endothelial PKR inhibits the phenotypic transformation of vascular smooth muscle (A) The diagram of co-culture system. (B) Representative immunoblots and densitometric analysis of α-SMA, SM22α, caldesmon, calponin, thrombospondin and osteopontin level in HASMCs that co-cultured with indicated HUVECs (n = 3). (C) Representative immunoblots and densitometric analysis of α-SMA, SM22α, caldesmon, calponin, thrombospondin and osteopontin level in HASMCs that co-cultured with indicated HUVECs (n = 3). (D) Representative immunoblots and densitometric analysis of α-SMA, SM22α, caldesmon, calponin, thrombospondin and osteopontin level in HASMCs that co-cultured with indicated HUVECs (n = 3). ∗ p< 0.05; ∗∗ p< 0.01; ∗∗∗ p< 0.001 (two-way ANOVA test). Data are represented as mean ± SEM.

Article Snippet: The membranes were blocked with 5% fat-free milk before incubation with primary antibodies specifically recognizing PKR (ab32052, 1:1000, Abcam), p-PKR (ab32036, 1:1000, Abcam), human GSDMD (ab210070, 1:1000, Abcam), rabbit anti-human N-GSDMD antibody (EPR20829-408) (ab215203, 1:1000, Abcam) and IL-1β (MAB201, 1:1000, R&D system), α-SMA (1:1000, Proteintech), SM22α (1:1000, Proteintech), calponin (1:1000, Proteintech), caldesmon (1:1000, Proteintech), thrombospondin (1:1000, Proteintech) and osteopontin (1:1000, Proteintech).

Techniques: Transformation Assay, Co-Culture Assay, Western Blot, Cell Culture

Journal: iScience

Article Title: PKR deficiency delays vascular aging via inhibiting GSDMD-mediated endothelial cell hyperactivation

doi: 10.1016/j.isci.2022.105909

Figure Lengend Snippet: Silencing endothelial PKR inhibits palmitic acid-induced endothelial cell hyperactivation and phenotypic transformation of vascular smooth muscle (A) Representative immunoblots and densitometric analysis of GSDMD, N-GSDMD, PKR and p -PKR level in siCon and PKR knockdown (siPKR) HUVECs stimulated with palmitic acid (n = 3). (B) LDH assay in the supernatants of siCon and PKR knockdown (siPKR) HUVECs stimulated with palmitic acid (n = 3). (C) Representative immunoblots and densitometric analysis of α-SMA, SM22α, caldesmon, calponin, thrombospondin and osteopontin level in HASMCs that co-cultured with indicated HUVECs (n = 3). ∗ p< 0.05; ∗∗ p< 0.01; ∗∗∗ p< 0.001 (two-way ANOVA test). Data are represented as mean ± SEM.

Article Snippet: The membranes were blocked with 5% fat-free milk before incubation with primary antibodies specifically recognizing PKR (ab32052, 1:1000, Abcam), p-PKR (ab32036, 1:1000, Abcam), human GSDMD (ab210070, 1:1000, Abcam), rabbit anti-human N-GSDMD antibody (EPR20829-408) (ab215203, 1:1000, Abcam) and IL-1β (MAB201, 1:1000, R&D system), α-SMA (1:1000, Proteintech), SM22α (1:1000, Proteintech), calponin (1:1000, Proteintech), caldesmon (1:1000, Proteintech), thrombospondin (1:1000, Proteintech) and osteopontin (1:1000, Proteintech).

Techniques: Transformation Assay, Western Blot, Knockdown, Lactate Dehydrogenase Assay, Cell Culture

Journal: iScience

Article Title: PKR deficiency delays vascular aging via inhibiting GSDMD-mediated endothelial cell hyperactivation

doi: 10.1016/j.isci.2022.105909

Figure Lengend Snippet:

Article Snippet: The membranes were blocked with 5% fat-free milk before incubation with primary antibodies specifically recognizing PKR (ab32052, 1:1000, Abcam), p-PKR (ab32036, 1:1000, Abcam), human GSDMD (ab210070, 1:1000, Abcam), rabbit anti-human N-GSDMD antibody (EPR20829-408) (ab215203, 1:1000, Abcam) and IL-1β (MAB201, 1:1000, R&D system), α-SMA (1:1000, Proteintech), SM22α (1:1000, Proteintech), calponin (1:1000, Proteintech), caldesmon (1:1000, Proteintech), thrombospondin (1:1000, Proteintech) and osteopontin (1:1000, Proteintech).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Staining, Lysis, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Isolation, Transfection, Negative Control, Software, Electron Microscopy