Journal: Oncology Research

Article Title: Discovery of Two Novel Pyrazole Derivatives as Anticancer Agents Targeting Tubulin Polymerization and MAPK Signaling Pathways

doi: 10.32604/or.2026.074945

Figure Lengend Snippet: Gene expression analysis induced by P3C.1 and P3C.2 in three different cell lines. ( A ) Heatmap of differentially expressed genes of MDA-MB-231, ( B ) Jurkat and ( C ) CEM cells treated with P3C.1 and P3C.2, or with solvent control (DMSO). Each column represents the expression values of the different treatments performed for 6 h for each corresponding cell line, and each row denotes data from one transcript. The color legend of each expression values (log2 fold change) is displayed on the right. ( D ) Differentially expressed genes; up-regulated (green bars) and down-regulated (red bars) obtained for each cell line and treatments, considering only transcripts with a log2 fold change of >1.0, and a p -adjusted value of <0.05.

Article Snippet: In addition, the RAMOS (ATCC, CRL-1596), CEM (ATCC, CCL-119), JURKAT (ATCC, TIB-152), NALM-6 (ATCC, CRL-3273), RPMI-8226, MM.1S, MM.1R, U266, Rec-1, JVM-13, A549 (ATCC, CRM-CCL-185), HCC70 (ATCC, CRL-2315), HCC1419 (ATCC, CRL-2326), T47D (ATCC, CRL-2865), KMS-11, and OVCAR-5 cell lines were grown in Roswell Park Memorial Institute-1640 (RPMI-1640) culture media (Cytiva-Hyclone, SH30027FS, UT, USA,) supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin.

Techniques: Gene Expression, Solvent, Control, Expressing

Journal: Oncology Research

Article Title: Discovery of Two Novel Pyrazole Derivatives as Anticancer Agents Targeting Tubulin Polymerization and MAPK Signaling Pathways

doi: 10.32604/or.2026.074945

Figure Lengend Snippet: P3C.1 and P3C.2 differentially expressed genes induced in common. ( A ) MDA-MB-231, ( B ) Jurkat, and CEM common DEGs identified for both compounds. ( C ) P3C.1 and P3C.2 induced 4 up-regulated genes in common (DUSP8, KLF6, KLF7 and BACH2) in MDA-MB-231, Jurkat and CEM cell lines.

Article Snippet: In addition, the RAMOS (ATCC, CRL-1596), CEM (ATCC, CCL-119), JURKAT (ATCC, TIB-152), NALM-6 (ATCC, CRL-3273), RPMI-8226, MM.1S, MM.1R, U266, Rec-1, JVM-13, A549 (ATCC, CRM-CCL-185), HCC70 (ATCC, CRL-2315), HCC1419 (ATCC, CRL-2326), T47D (ATCC, CRL-2865), KMS-11, and OVCAR-5 cell lines were grown in Roswell Park Memorial Institute-1640 (RPMI-1640) culture media (Cytiva-Hyclone, SH30027FS, UT, USA,) supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin.

Techniques:

Journal: Oncology Research

Article Title: Discovery of Two Novel Pyrazole Derivatives as Anticancer Agents Targeting Tubulin Polymerization and MAPK Signaling Pathways

doi: 10.32604/or.2026.074945

Figure Lengend Snippet: Ingenuity pathways analysis of genes commonly found in P3C.1 and P3C.2-treated MDA-MB231, JURKAT, and CEM cells, identified canonical pathways implicating these genes in kinase-mediated signal transduction (see green asterisks * ), including RAF-independent MAPK1/3 activation, RAF/MAP kinase cascade, Protein Kinase A Signaling, and the SAPK/JNK signaling pathway, as well as cell signaling processes (see blue asterisks * ), and membrane dynamics (see blue asterisks * ). The figure shown was recreated from the original IPA histogram to enlarge features and improve legibility.

Article Snippet: In addition, the RAMOS (ATCC, CRL-1596), CEM (ATCC, CCL-119), JURKAT (ATCC, TIB-152), NALM-6 (ATCC, CRL-3273), RPMI-8226, MM.1S, MM.1R, U266, Rec-1, JVM-13, A549 (ATCC, CRM-CCL-185), HCC70 (ATCC, CRL-2315), HCC1419 (ATCC, CRL-2326), T47D (ATCC, CRL-2865), KMS-11, and OVCAR-5 cell lines were grown in Roswell Park Memorial Institute-1640 (RPMI-1640) culture media (Cytiva-Hyclone, SH30027FS, UT, USA,) supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin.

Techniques: Transduction, Activation Assay, Membrane

Journal: Oncology Research

Article Title: Discovery of Two Novel Pyrazole Derivatives as Anticancer Agents Targeting Tubulin Polymerization and MAPK Signaling Pathways

doi: 10.32604/or.2026.074945

Figure Lengend Snippet: Connectivity map analyses revealed strong connectivity of P3C compounds with tubulin inhibitors. The top 20 perturbagens identified using P3C.1 and P3C.2 genes in common for ( A ) MDA-MB-231, and ( B ) Jurkat, and CEM cell lines. Tau scores of 90 or above have been previously recognized as strong and suitable for further investigation ( https://clue.io ).

Article Snippet: In addition, the RAMOS (ATCC, CRL-1596), CEM (ATCC, CCL-119), JURKAT (ATCC, TIB-152), NALM-6 (ATCC, CRL-3273), RPMI-8226, MM.1S, MM.1R, U266, Rec-1, JVM-13, A549 (ATCC, CRM-CCL-185), HCC70 (ATCC, CRL-2315), HCC1419 (ATCC, CRL-2326), T47D (ATCC, CRL-2865), KMS-11, and OVCAR-5 cell lines were grown in Roswell Park Memorial Institute-1640 (RPMI-1640) culture media (Cytiva-Hyclone, SH30027FS, UT, USA,) supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin.

Techniques:

Journal: Nature Communications

Article Title: Activity-dependent isomerization of Kv4.2 by Pin1 regulates cognitive flexibility

doi: 10.1038/s41467-020-15390-x

Figure Lengend Snippet: a Pin1 co-immunoprecipitated with Kv4.2 in mouse brain lysates. Forebrain lysates from WT and Kv4.2 KO were immunoprecipitated with mouse (ms) or rabbit (rb) anti-Kv4.2 antibodies. Both total lysates and immunoprecipitates were blotted with anti-Kv4.2 or Pin1 antibodies. Data from three independent experiments. b Cultured hippocampal neurons (DIV 10) were immunostained with anti-Pin1 along with anti-Kv4.2. Pin1 co-localized with Kv4.2, indicated with arrows. Scale bars: 20 μm top panels, 5 μm bottom. Data from four coverslips in two independent experiments. c , Pin1 mutants reduced Pin1-Kv4.2 binding. Myc-Kv4.2 was co-transfected alongside HA-Pin1 with or without WW (W34A) or PPIase domain (R68, R69A) point mutants into HEK-293T cells. Kv4.2 was immunoprecipitated from detergent lysates with anti-Myc antibody. Samples were analyzed by western blotting with anti-HA and anti-Myc antibodies. n = 3 each group. d Alignment of Kv4.2 C-terminal sequences from various species. The putative Pin1 binding site is conserved. Bold residues show preferred Pin1 binding context. e Pin1 selectively binds to the phosoho-T607-containing Kv4.2 peptide. Synthetic Kv4.2-peptides were conjugated to Affi-Gel 15 Sepharose beads and incubated with lysate from HA-Pin1 transfected HEK-293T cells. n = 4 each group. f Kv4.2 T607A mutation significantly reduced Pin1 binding. HA-Pin1 and Myc-Kv4.2 mutants were co-transfected into HEK-293T cells. Pin1 co-immunoprecipitation with Kv4.2 was assayed. Kv4.2 T607 is required for Pin1 binding. n = 3 each group. g Molecular modeling of Kv4.2 phospho-peptide binding to Pin1. h Highlight of Kv4.2 pT602 peptide binding to the Pin1 WW domain. i Highlight of Kv4.2 pT607 peptide binding to the Pin1 PPIase domain. j Dose-dependent proteolysis of Kv4.2 by subtilisin. Asterisk, non-specific bands; arrowhead, 47kD band; arrow, 33kD band. Data repeated in two independent experiments. k Pin1 blocked Kv4.2 subtilisin digestion while Pin1C113S (an isomerase dead mutant) did not. Pin1 block was lost when Kv4.2 was dephosphorylated by Lambda protein phosphatase (PP). Quantification of the 47kD degradation fragment. n = 4 each group. Data was repeated in four independent experiments. Data are presented as mean ± SEM, * p < 0.05, *** p < 0.001, Paired t -tests.

Article Snippet: Mouse anti-Kv4.2 (NeuroMab, 75-016) was used at 1:2000 for western blot, 1:200 for immunostaining, Rabbit anti-Kv4.2 (Sigma, P0233) was used at 1:2000 for western blot, rabbit anti-Kv4.2 (Sigma, HPA029068) was used at 1:200 for staining, pT602 (Santa Cruz, SC-16983-R) was used at 1:1000 for western blot, pT607 (Santa Cruz, SC-22254-R) was used at 1:500 for western blot, Pin1 (Santa Cruz, SC-46660) was used at 1:100 for staining, 1:1000 for western blot, Pin1 (Millipore, 07-091) was used at 1:3000 for western blot, p38 (Cell Signaling, 9212 s) was used at 1:1000 for western blot, p-p38 (Cell Signaling, 4511 s) at 1:1000 for western blot, DPP6 (Abcam, 41811) was used at 1:2000 for western blot, Myc (Millipore, 05-419) was used at 1:10000 for western blot, HA(Santa Cruz, SC-805) was used at 1:1000 for western blot, Actin (Sigma, A-1978) was used at 1:10000 for western blot; Alexa Fluor 488 goat anti-mouse (Invitrogen, A-11029) was used at 1:500; Alexa Fluor 488 goat anti-rabbit (Invitrogen, A-11034) was used at 1:500; Alexa Fluor 555 goat anti-mouse (Invitrogen, A-21424) was used at 1:500; Alexa Fluor 555 goat anti-rabbit (Invitrogen, A-21429) was used at 1:500; Alexa Fluor 680 goat anti-mouse (Invitrogen, A-21057) was used at 1:10000; Alexa Fluor 680 goat anti-rabbit (Invitrogen, A-21076) was used at 1:10000; IRDye 800CW goat anti-mouse (Licor, 926-32210) was used at 1:10000, IRDye 800CW goat anti-rabbit (Licor, 926-32211) was used at 1:10000.

Techniques: Immunoprecipitation, Cell Culture, Binding Assay, Transfection, Western Blot, Incubation, Mutagenesis, Blocking Assay

Journal: Nature Communications

Article Title: Activity-dependent isomerization of Kv4.2 by Pin1 regulates cognitive flexibility

doi: 10.1038/s41467-020-15390-x

Figure Lengend Snippet: a Enriched novel environment (EE, 1 h) induces phosphorylation of Kv4.2 at Thr607 but not Thr602 in mouse hippocampus. n = 5 in each group. T -test, * p < 0.05. b Kainic acid-induced seizure (25 mg/kg, i.p., 15 min) induces phosphorylation of Kv4.2 at Thr607 but not Thr602 in mouse hippocampus. n = 4 in each group. T -test, *** p < 0.001. c PTZ-induced seizure (50 mg/kg, i.p., 15 min) induces phosphorylation of Kv4.2 at Thr607 and Thr602 in mouse hippocampus. n = 4 in each group. * p < 0.05, T -test, ** p < 0.01. d PTZ-induced seizure increases Pin1 binding to Kv4.2. GST or GST-Pin1-linked beads were incubated with brain lysates from mice subjected to saline or PTZ administration. n = 5 in each group. T -test, ** p < 0.01. e Mouse brain lysates from WT mice w or w/o PTZ administration (50 mg/kg, i.p., 15 min) were incubated with excess anti-Kv4.2, anti-Kv4.2-pT602 or anti-Kv4.2-pT607 antibodies. Immunoprecipitation (IP) samples were blotted with Kv4.2 antibody. In WT mouse brains, pT607 Kv4.2 is almost half as abundant as pT602. However, PTZ administration increased the amount of pT607 until it reached a similar level as pT602. n = 4 for each group. T -test, ** p < 0.01. f Mouse brain lysates from WT mice were incubated with excess anti-Kv4.2-pT602 or normal IgG antibodies. Immunoprecipitation (IP) samples were blotted with anti-Kv4.2 pT607 antibody. pT602 and pT607 dual phosphorylation was observed in mouse brain. Data was repeated in two independent experiments. g Mouse brain lysates from WT mice w or w/o PTZ administration (50 mg/kg, i.p., 15 min) were incubated with excess anti-Kv4.2-pT607 antibody. IP samples were blotted with anti-Kv4.2 pT602 antibody. PTZ-induced seizure increases the dual phosphorylation of T602 and T607 in mouse brain. Data was repeated in two independent experiments. Data are presented as mean ± SEM.

Article Snippet: Mouse anti-Kv4.2 (NeuroMab, 75-016) was used at 1:2000 for western blot, 1:200 for immunostaining, Rabbit anti-Kv4.2 (Sigma, P0233) was used at 1:2000 for western blot, rabbit anti-Kv4.2 (Sigma, HPA029068) was used at 1:200 for staining, pT602 (Santa Cruz, SC-16983-R) was used at 1:1000 for western blot, pT607 (Santa Cruz, SC-22254-R) was used at 1:500 for western blot, Pin1 (Santa Cruz, SC-46660) was used at 1:100 for staining, 1:1000 for western blot, Pin1 (Millipore, 07-091) was used at 1:3000 for western blot, p38 (Cell Signaling, 9212 s) was used at 1:1000 for western blot, p-p38 (Cell Signaling, 4511 s) at 1:1000 for western blot, DPP6 (Abcam, 41811) was used at 1:2000 for western blot, Myc (Millipore, 05-419) was used at 1:10000 for western blot, HA(Santa Cruz, SC-805) was used at 1:1000 for western blot, Actin (Sigma, A-1978) was used at 1:10000 for western blot; Alexa Fluor 488 goat anti-mouse (Invitrogen, A-11029) was used at 1:500; Alexa Fluor 488 goat anti-rabbit (Invitrogen, A-11034) was used at 1:500; Alexa Fluor 555 goat anti-mouse (Invitrogen, A-21424) was used at 1:500; Alexa Fluor 555 goat anti-rabbit (Invitrogen, A-21429) was used at 1:500; Alexa Fluor 680 goat anti-mouse (Invitrogen, A-21057) was used at 1:10000; Alexa Fluor 680 goat anti-rabbit (Invitrogen, A-21076) was used at 1:10000; IRDye 800CW goat anti-mouse (Licor, 926-32210) was used at 1:10000, IRDye 800CW goat anti-rabbit (Licor, 926-32211) was used at 1:10000.

Techniques: Phospho-proteomics, Binding Assay, Incubation, Saline, Immunoprecipitation

Journal: Nature Communications

Article Title: Activity-dependent isomerization of Kv4.2 by Pin1 regulates cognitive flexibility

doi: 10.1038/s41467-020-15390-x

Figure Lengend Snippet: a P38 phosphorylates Kv4.2 at Thr602 and Thr607. Kv4.2 and p38α constructs (agf: p38 kinase dead mutant) were co-transfected into HEK-293T cells. Lysates were analyzed by western blotting with anti-pT602 and anti-pT607 specific antibodies. n = 15 for ctl, 9 for p38 and p38 agf. T -test, *** p < 0.001. b p38 binds to Kv4.2. Kv4.2 and p38α were co-transfected into HEK-293T cells. Detergent lysates were incubated with anti-Myc antibody and analyzed by western blotting with anti-Flag and anti-Myc antibodies. Data was repeated in two independent experiments. c , Enriched novel environment activates p38 in mouse hippocampus. n = 6 in each group. T -test, * p < 0.05. d KA-induced seizure activates p38 in mouse hippocampus. n = 8 in each group. T -test, ***p < 0.001. e PTZ-induced seizure activates p38 in mouse hippocampus. n = 6 for ctl and 5 for PTZ. T -test, *** p < 0.001. f SB203580, a potent p38 inhibitor (20 mg/kg, i.p., 15 min) blocked PTZ-induced phosphorylation of Kv4.2 T607 in mouse hippocampus. n = 4 in each group. T -test, ** p < 0.01. g SL327, a selective MEK inhibitor (30 mg/kg, i.p., 15 min) did not block PTZ-induced phosphorylation of Kv4.2 T607 in mouse hippocampus. n = 7 in each group. * p < 0.05, ** p < 0.01. t -test. Data are presented as mean ± SEM.

Article Snippet: Mouse anti-Kv4.2 (NeuroMab, 75-016) was used at 1:2000 for western blot, 1:200 for immunostaining, Rabbit anti-Kv4.2 (Sigma, P0233) was used at 1:2000 for western blot, rabbit anti-Kv4.2 (Sigma, HPA029068) was used at 1:200 for staining, pT602 (Santa Cruz, SC-16983-R) was used at 1:1000 for western blot, pT607 (Santa Cruz, SC-22254-R) was used at 1:500 for western blot, Pin1 (Santa Cruz, SC-46660) was used at 1:100 for staining, 1:1000 for western blot, Pin1 (Millipore, 07-091) was used at 1:3000 for western blot, p38 (Cell Signaling, 9212 s) was used at 1:1000 for western blot, p-p38 (Cell Signaling, 4511 s) at 1:1000 for western blot, DPP6 (Abcam, 41811) was used at 1:2000 for western blot, Myc (Millipore, 05-419) was used at 1:10000 for western blot, HA(Santa Cruz, SC-805) was used at 1:1000 for western blot, Actin (Sigma, A-1978) was used at 1:10000 for western blot; Alexa Fluor 488 goat anti-mouse (Invitrogen, A-11029) was used at 1:500; Alexa Fluor 488 goat anti-rabbit (Invitrogen, A-11034) was used at 1:500; Alexa Fluor 555 goat anti-mouse (Invitrogen, A-21424) was used at 1:500; Alexa Fluor 555 goat anti-rabbit (Invitrogen, A-21429) was used at 1:500; Alexa Fluor 680 goat anti-mouse (Invitrogen, A-21057) was used at 1:10000; Alexa Fluor 680 goat anti-rabbit (Invitrogen, A-21076) was used at 1:10000; IRDye 800CW goat anti-mouse (Licor, 926-32210) was used at 1:10000, IRDye 800CW goat anti-rabbit (Licor, 926-32211) was used at 1:10000.

Techniques: Construct, Mutagenesis, Transfection, Western Blot, Incubation, Phospho-proteomics, Blocking Assay

Journal: Nature Communications

Article Title: Activity-dependent isomerization of Kv4.2 by Pin1 regulates cognitive flexibility

doi: 10.1038/s41467-020-15390-x

Figure Lengend Snippet: a , b Pin1 inhibitor PiB (4 µM) reduces pyramidal cell excitability in WT mouse hippocampal brain slices. a Current steps at 100 pA and 200 pA result in reduced firing frequency with PiB application (teal) ( n = 13) relative to vehicle (green) ( n = 14). This reduction is rescued by co-application of AmmTX3 (250 nM) (blue) ( n = 7). Scale: 30 mV / 250 ms. b , PiB significantly reduces AP firing frequency relative to vehicle in response to 100, 150, and 200 pA somatic current injections. This reduction is rescued by AmmTX3 application. Two-way ANOVA, * p < 0.05, ** p < 0.01. c , d Mutation of Kv4.2 T607 Pin1 binding site phenocopies pharmacological inhibition of Pin1 in WT. c Pyramidal cells from Kv4.2TA slices display reduced firing frequency relative to WT over a range of increasing current injections. Scale: 30 mV/250 ms. d AP firing frequency is significantly reduced after 150 and 200 pA somatic current injections in Kv4.2TA ( n = 22) relative to WT ( n = 20), two-way ANOVA, ** p < 0.01, ***p < 0.001. e , f Pin1 inhibition with PiB has no effect in Kv4.2TA mice. e Pyramidal cells from Kv4.2TA slices display similar AP firing patterns when treated with vehicle ( n = 14) and PiB ( n = 14). Scale: 30 mV/250 ms. f No significant changes are observed in Kv4.2TA pyramidal cell AP firing frequency with PiB exposure, Two-Way ANOVA, p > 0.05. g Brain slicing and recovery activates p38 and increases pT607 of Kv4.2. Adult mouse brains were either sliced as for electrophysiological recordings or dissected as for biochemical assays. Same brain regions were used. n = 4 for ctl and 3 for slicing. T -test. * p < 0.05, *** p < 0.001. Data are presented as mean ± SEM.

Article Snippet: Mouse anti-Kv4.2 (NeuroMab, 75-016) was used at 1:2000 for western blot, 1:200 for immunostaining, Rabbit anti-Kv4.2 (Sigma, P0233) was used at 1:2000 for western blot, rabbit anti-Kv4.2 (Sigma, HPA029068) was used at 1:200 for staining, pT602 (Santa Cruz, SC-16983-R) was used at 1:1000 for western blot, pT607 (Santa Cruz, SC-22254-R) was used at 1:500 for western blot, Pin1 (Santa Cruz, SC-46660) was used at 1:100 for staining, 1:1000 for western blot, Pin1 (Millipore, 07-091) was used at 1:3000 for western blot, p38 (Cell Signaling, 9212 s) was used at 1:1000 for western blot, p-p38 (Cell Signaling, 4511 s) at 1:1000 for western blot, DPP6 (Abcam, 41811) was used at 1:2000 for western blot, Myc (Millipore, 05-419) was used at 1:10000 for western blot, HA(Santa Cruz, SC-805) was used at 1:1000 for western blot, Actin (Sigma, A-1978) was used at 1:10000 for western blot; Alexa Fluor 488 goat anti-mouse (Invitrogen, A-11029) was used at 1:500; Alexa Fluor 488 goat anti-rabbit (Invitrogen, A-11034) was used at 1:500; Alexa Fluor 555 goat anti-mouse (Invitrogen, A-21424) was used at 1:500; Alexa Fluor 555 goat anti-rabbit (Invitrogen, A-21429) was used at 1:500; Alexa Fluor 680 goat anti-mouse (Invitrogen, A-21057) was used at 1:10000; Alexa Fluor 680 goat anti-rabbit (Invitrogen, A-21076) was used at 1:10000; IRDye 800CW goat anti-mouse (Licor, 926-32210) was used at 1:10000, IRDye 800CW goat anti-rabbit (Licor, 926-32211) was used at 1:10000.

Techniques: Mutagenesis, Binding Assay, Inhibition

Journal: Nature Communications

Article Title: Activity-dependent isomerization of Kv4.2 by Pin1 regulates cognitive flexibility

doi: 10.1038/s41467-020-15390-x

Figure Lengend Snippet: a In WT mice, stimulations such as seizure and exposure to a novel environment trigger the phosphorylation of Kv4.2 at T607, which allows Pin1 binding to pT602 and pT607 which subsequently isomerizes the pT607-P bond. This process changes the conformation of Kv4.2, which dissociates the Kv4.2-DPP6 complex and increases neuronal excitability and cognitive inflexibility. b In Kv4.2 TA mice, the 607 site is no longer phosphorylatable so that Pin1’s effect on Kv4.2 is abolished. The Kv4.2-DPP6 complex is stable, neuronal excitability is reduced, and cognitive flexibility is improved.

Article Snippet: Mouse anti-Kv4.2 (NeuroMab, 75-016) was used at 1:2000 for western blot, 1:200 for immunostaining, Rabbit anti-Kv4.2 (Sigma, P0233) was used at 1:2000 for western blot, rabbit anti-Kv4.2 (Sigma, HPA029068) was used at 1:200 for staining, pT602 (Santa Cruz, SC-16983-R) was used at 1:1000 for western blot, pT607 (Santa Cruz, SC-22254-R) was used at 1:500 for western blot, Pin1 (Santa Cruz, SC-46660) was used at 1:100 for staining, 1:1000 for western blot, Pin1 (Millipore, 07-091) was used at 1:3000 for western blot, p38 (Cell Signaling, 9212 s) was used at 1:1000 for western blot, p-p38 (Cell Signaling, 4511 s) at 1:1000 for western blot, DPP6 (Abcam, 41811) was used at 1:2000 for western blot, Myc (Millipore, 05-419) was used at 1:10000 for western blot, HA(Santa Cruz, SC-805) was used at 1:1000 for western blot, Actin (Sigma, A-1978) was used at 1:10000 for western blot; Alexa Fluor 488 goat anti-mouse (Invitrogen, A-11029) was used at 1:500; Alexa Fluor 488 goat anti-rabbit (Invitrogen, A-11034) was used at 1:500; Alexa Fluor 555 goat anti-mouse (Invitrogen, A-21424) was used at 1:500; Alexa Fluor 555 goat anti-rabbit (Invitrogen, A-21429) was used at 1:500; Alexa Fluor 680 goat anti-mouse (Invitrogen, A-21057) was used at 1:10000; Alexa Fluor 680 goat anti-rabbit (Invitrogen, A-21076) was used at 1:10000; IRDye 800CW goat anti-mouse (Licor, 926-32210) was used at 1:10000, IRDye 800CW goat anti-rabbit (Licor, 926-32211) was used at 1:10000.

Techniques: Phospho-proteomics, Binding Assay