Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: A. In vitro phosphorylation of brain homogenate by recombinant active JNK1 revealed phosphorylation of GIT1 on S371 and S692. Mass spectrometry-identified sequences are shown for GIT1 (accession NP_001078923.1). Β. Representative autoradiograph and Coomassie Brilliant Blue (CBB) stained gel image are shown for recombinant GST-GIT1- wild-type (WT), -S371A, -S692A or S371A/S692A (SAA) variants after phosphorylation by GST-JNK1. JNK1 phosphorylated GIT1-WT and GIT1–S692A but not GIT1-S371A, indicating that S371 is the preferred JNK1 phosphorylation site. Phosphorylation of GIT1 by JNK1 was prevented by JNK inhibitor 10 µM SP600125. C. Quantitative data from 4 repeats of experiment shown in B . D. Domain map of human GIT1 (UniProt identifier: Q9Y2X7-3) shows predicted JNK1 phosphorylation sites (yellow circles). Abbreviations: Arf GTPase activating protein domain (GAP), 3x Ankyrin repeats (ANK), Spa-Homology domain (SHD), Synapse Localization Domain (SLD) and Paxillin Binding Domain (PBD). E. Quantitative LC-MS/MS data of GIT1-S371 phosphorylation in wild-type (WT) and Jnk1-/- whole brains. F. Quantitative LC-MS/MS data of GIT1-S371 phosphorylation in brains from DJNKI-1- infused adult mice. G. Active Arf1 was detected from pull downs using GST-GAT-GGA3 immobilized on glutathione beads. Cells expressed GIT1-variants together with Arf1-WT or constitutively active Arf1-Q71L. H. Quantitative data from 3 repeats of experiment described in G. Phosphorylation-site mutants of GIT1 did not alter Arf1 activity. Error bars represent standard error of the mean (SEM) and P-values were determined using Student’s t test.

Article Snippet: For in vitro phosphorylation assay, pEBG-JNK1α1 and pEGFP-MEKK1Δ(1174-1493) have been previously described ( ). pmRuby2-Lifeact7 was from Michael Davidson’s lab via Addgene, pEGFP-Dynamin 2-WT and pEGFP-Dynamin 2-K44A was from Pietro De Camilli. pcDNA3-HA-Arf1 (Addgene plasmid # 10830) and pcDNA3-HA-Arf1-ActQ71L (Addgene plasmid # 10832) were generated by Thomas Roberts. pGEX-5x-1-VHS GAT-GGA3 was a gift from Juan S. Bonifacino. pEGFP-PXN-WT was described previously ( ).

Techniques: In Vitro, Recombinant, Mass Spectrometry, Autoradiography, Staining, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal: International Journal of Biological Sciences

Article Title: A Sonic Hedgehog-Gli-Bmi1 signaling pathway plays a critical role in p27 deficiency induced bone anabolism

doi: 10.7150/ijbs.65954

Figure Lengend Snippet: Deletion of Bmi1 blocks p27 deficiency-induced osteoblastic bone formation in vivo . (A, C) Representative micro-CT scans of 3-dimensional longitudinal reconstructions of proximal ends of tibiae and midshaft diaphysis from 4-week-old WT, p27 -/- , Bmi1 -/- and p27 -/- Bmi1 -/- mice. (B) Trabecular bone volume relative to tissue volume (BV/TV, %). (D) Cortical bone volume (mm 3 ). Representative micrographs of paraffin-embedded sections of tibias from 4-week-old WT, p27 -/- , Bmi1 -/- and p27 -/- Bmi1 -/- mice: (E) staining with H&E and (F) the number of osteoblasts per mm 2 tissue area (N.Ob/T.Ar, #/mm 2 ), (G) immunohistochemical staining for type I collagen (Col-I) and (H) Col-I-positive areas as a percent of the tissue area (%), (I) histochemical staining for TRAP and (J) osteoclast surface/bone surface (Oc.S/BS, %). Real-time RT-PCR analyses of long bone extracts for the expression of (K) ALP , (L) Runx2 , (M) Col-I and (N) OCN . Messenger RNA expression assessed by RT-PCR was calculated as a ratio relative to the GAPDH mRNA level and expressed relative to WT control. Values are mean ± s. e. m. of 5 determinations per group. *: P <0.05; **: P <0.01 compared with WT mice. ###: P <0.001 compared with p27 -/- mice.

Article Snippet: Immunohistochemistry was performed with the primary antibodies against Shh (ab135240; Abcam, Cambridge, MA, USA), Bmi1 (10832-1-AP; Proteintech, USA), and Gli2 (18989-1-AP; Proteintech, USA), Col-I (34710; Abcam, Cambridge, MA, USA).

Techniques: In Vivo, Micro-CT, Staining, Immunohistochemical staining, Quantitative RT-PCR, Expressing, RNA Expression, Reverse Transcription Polymerase Chain Reaction, Control