Journal: Open Medicine

Article Title: lncRNA ZFAS1 promotes intervertebral disc degeneration by upregulating AAK1

doi: 10.1515/med-2022-0530

Figure Lengend Snippet: The sequences of primers for RT-qPCR

Article Snippet: Western blotting was performed as previously recorded [ ] with primary antibodies: Bax (ab32503; 1:1,000, Abcam), Bcl-2 (ab182858; 1:2,000), cleaved caspase-3 (9661; 1:1,000, Cell Signal Technology), cleaved caspase-9 (9508; 1:1,000), AAK1 (61527; 1:1,000), Collagen II (ab34712; 1:1,000), Aggrecan (MA5-42646; 1:1,000, Invitrogen), MMP-3 (ab52915; 1:1,000), MMP-13 (a39012; 1:3,000), and GAPDH (ab181602; 1:10,000).

Techniques: Sequencing

Journal: Open Medicine

Article Title: lncRNA ZFAS1 promotes intervertebral disc degeneration by upregulating AAK1

doi: 10.1515/med-2022-0530

Figure Lengend Snippet: AAK1 is targeted by miR-4711-5p. (a) Ten putative mRNAs capable of binding to miR-4711-5p were found in TargetScan database. (b) The mRNA expression in NP cells treated with 10 ng/mL IL-1β. (c) Binding sequence of miR-4711-5p complementary to AAK1. (d) Overexpression efficiency of pcDNA/ZFAS1 in NP cells. (e) Luciferase activity in HEK293T cells transfected with AAK1-Wt or AAK1-Mut reporter vector together with the indicated plasmids. (f and g) RT-qPCR and western blotting analysis of AAK1 expression in NP cells transfected with NC mimics, miR-4711-5p mimics, and miR-4711-5p mimics + pcDNA/ZFAS1. (h) RT-qPCR analysis of AAK1 expression in NP tissues of IDD patients. (i) Correlation between AAK1 and miR-4711-5p expression in NP tissues of IDD patients. ** p < 0.01, *** p < 0.001.

Article Snippet: Western blotting was performed as previously recorded [ ] with primary antibodies: Bax (ab32503; 1:1,000, Abcam), Bcl-2 (ab182858; 1:2,000), cleaved caspase-3 (9661; 1:1,000, Cell Signal Technology), cleaved caspase-9 (9508; 1:1,000), AAK1 (61527; 1:1,000), Collagen II (ab34712; 1:1,000), Aggrecan (MA5-42646; 1:1,000, Invitrogen), MMP-3 (ab52915; 1:1,000), MMP-13 (a39012; 1:3,000), and GAPDH (ab181602; 1:10,000).

Techniques: Binding Assay, Expressing, Sequencing, Over Expression, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot

Journal: Open Medicine

Article Title: lncRNA ZFAS1 promotes intervertebral disc degeneration by upregulating AAK1

doi: 10.1515/med-2022-0530

Figure Lengend Snippet: ZFAS1 facilitates NP cell apoptosis and ECM degradation by AAK1. NP cells were then treated with IL-1β + sh-NC, IL-1β + sh-ZFAS1, IL-1β + pcDNA, and IL-1β + pcDNA/AAK1M, respectively. (a) AAK1 expression in NP cells. (b and c) EdU assay of cell proliferation in each group. (d and e) Flow cytometry analysis of cell apoptosis in each group. (f–i) Western blotting analysis of the expression of Bax, Bcl-2, Collagen II, Aggrecan, MMP-3, and MMP-13 in each group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Western blotting was performed as previously recorded [ ] with primary antibodies: Bax (ab32503; 1:1,000, Abcam), Bcl-2 (ab182858; 1:2,000), cleaved caspase-3 (9661; 1:1,000, Cell Signal Technology), cleaved caspase-9 (9508; 1:1,000), AAK1 (61527; 1:1,000), Collagen II (ab34712; 1:1,000), Aggrecan (MA5-42646; 1:1,000, Invitrogen), MMP-3 (ab52915; 1:1,000), MMP-13 (a39012; 1:3,000), and GAPDH (ab181602; 1:10,000).

Techniques: Expressing, EdU Assay, Flow Cytometry, Western Blot

Journal: Open Medicine

Article Title: lncRNA ZFAS1 promotes intervertebral disc degeneration by upregulating AAK1

doi: 10.1515/med-2022-0530

Figure Lengend Snippet: ZFAS1 knockdown alleviates IDD in vivo . (a) Hematoxylin–eosin staining of intervertebral disc tissues of mice. N = 10. (b) Histological score in each group. N = 10. (c–f) Western blotting analysis of the expression of Bax, Bcl-2, Collagen II, Aggrecan, MMP-3, and MMP-13 in intervertebral disc tissues of mice. N = 10. (g and h) RT-qPCR analysis of the expression of ZFAS1 and AAK1 in intervertebral disc tissues of mice. N = 10. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Western blotting was performed as previously recorded [ ] with primary antibodies: Bax (ab32503; 1:1,000, Abcam), Bcl-2 (ab182858; 1:2,000), cleaved caspase-3 (9661; 1:1,000, Cell Signal Technology), cleaved caspase-9 (9508; 1:1,000), AAK1 (61527; 1:1,000), Collagen II (ab34712; 1:1,000), Aggrecan (MA5-42646; 1:1,000, Invitrogen), MMP-3 (ab52915; 1:1,000), MMP-13 (a39012; 1:3,000), and GAPDH (ab181602; 1:10,000).

Techniques: In Vivo, Staining, Western Blot, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Multiplexed profiling of kinase interactomes quantifies cellular network plasticity

doi: 10.1101/2021.09.14.460283

Figure Lengend Snippet: A) Kinome dendrogram overlayed with FOCUS cell kinases whose PPIs significantly change in response to AXL RNAi knockdown (BH-FDR < 0.05, n = 5). The PPI with the highest fold-change was used for potting. B) Ki-CCA interaction network of high confidence interactors (r > 0.6) for the AAK1/BMP2K kinase group identified across the 18-cell line panel. C) Abundance differences of AAK1/BMP2K network components between FOCUS WT/ AXL RNAi cells, and mesenchymal- like and epithelial-like cells of a 17 HCC line panel (kinobead/LC-MS data). * t-test significant, BH-FDR < 0.05. D) Abundance differences of AAK1/BMP2K complex components between human HCC tumor tissue and normal adjacent liver (kinobead/LC-MS data). * t-test significant, BH-FDR < 0.05.

Article Snippet: Antibodies used were anti-E-cadherin (24E10, Cell Signaling Technology, CST, Cat # 3195), anti-AXL (C89E7, CST, Cat # 8661), anti-Snail (C15D3, CST, Cat # 3879), anti-ZEB1 (E2G6Y, CST, Cat # 70512), anti-CD44 (E7K2Y, CST, Cat # 37259), anti-GAPDH HRP conjugate (D16H11, CST, Cat # 8884), anti-AAK1 (E8M3P, CST, Cat # 61527), anti-RALBP1 (D87H8, CST, Cat # 5739), and anti-REPS1 (D6F4, CST, Cat # 6404).

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Multiplexed profiling of kinase interactomes quantifies cellular network plasticity

doi: 10.1101/2021.09.14.460283

Figure Lengend Snippet: A) Kinobead/LC-MS soluble competition experiment using 1 µM of the selective AAK1 inhibitor LB-935509. Members of the AAK1/BMP2K PPI network are highlighted. B) Co-IP-MS experiments in FOCUS cells using antibodies targeting AAK1, RALBP1, and REPS1 validate the AAK1 complex’ identity. C) Western blot analysis of EMT markers in SNU387 and FOCUS RNAi lines. For analysis of the SKHep1 and SNU761 lines see . D) Kinobead/LC-MS profiling data of AAK1 complex RNAi lines showing that NF-kB and EMT kinases consistently decrease in expression in three out of four cell line models. E) Drug screen results demonstrating that AAK1 complex RNAi lines are up to 18-fold more sensitive to the CHEK1 and 2 inhibitors AZD-7762 and CHIR-124. F) Kinobead/LC-MS profiling from SKHep1 and SNU387 lines showing that AAK1 complex RNAi activates cell cycle-related kinases.

Article Snippet: Antibodies used were anti-E-cadherin (24E10, Cell Signaling Technology, CST, Cat # 3195), anti-AXL (C89E7, CST, Cat # 8661), anti-Snail (C15D3, CST, Cat # 3879), anti-ZEB1 (E2G6Y, CST, Cat # 70512), anti-CD44 (E7K2Y, CST, Cat # 37259), anti-GAPDH HRP conjugate (D16H11, CST, Cat # 8884), anti-AAK1 (E8M3P, CST, Cat # 61527), anti-RALBP1 (D87H8, CST, Cat # 5739), and anti-REPS1 (D6F4, CST, Cat # 6404).

Techniques: Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: Multiplexed profiling of kinase interactomes quantifies cellular network plasticity

doi: 10.1101/2021.09.14.460283

Figure Lengend Snippet: A) qPCR analysis of AAK1 complex RNAi lines, validating successful knockdown. B) Kinobead/LC-MS analysis of AAK1 complex RNAi lines, validating successful knockdown. C) Immunoblot analysis of AAK1 complex RNAi lines, validating successful knockdown. REPS2 blots not shown because the antibody used is likely not specific.

Article Snippet: Antibodies used were anti-E-cadherin (24E10, Cell Signaling Technology, CST, Cat # 3195), anti-AXL (C89E7, CST, Cat # 8661), anti-Snail (C15D3, CST, Cat # 3879), anti-ZEB1 (E2G6Y, CST, Cat # 70512), anti-CD44 (E7K2Y, CST, Cat # 37259), anti-GAPDH HRP conjugate (D16H11, CST, Cat # 8884), anti-AAK1 (E8M3P, CST, Cat # 61527), anti-RALBP1 (D87H8, CST, Cat # 5739), and anti-REPS1 (D6F4, CST, Cat # 6404).

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot

Journal: bioRxiv

Article Title: Multiplexed profiling of kinase interactomes quantifies cellular network plasticity

doi: 10.1101/2021.09.14.460283

Figure Lengend Snippet: Immunoblot analysis of EMT marker expression in AAK1 complex RNAi cell lines.

Article Snippet: Antibodies used were anti-E-cadherin (24E10, Cell Signaling Technology, CST, Cat # 3195), anti-AXL (C89E7, CST, Cat # 8661), anti-Snail (C15D3, CST, Cat # 3879), anti-ZEB1 (E2G6Y, CST, Cat # 70512), anti-CD44 (E7K2Y, CST, Cat # 37259), anti-GAPDH HRP conjugate (D16H11, CST, Cat # 8884), anti-AAK1 (E8M3P, CST, Cat # 61527), anti-RALBP1 (D87H8, CST, Cat # 5739), and anti-REPS1 (D6F4, CST, Cat # 6404).

Techniques: Western Blot, Marker, Expressing