Anti gapdh

Dimethyl sulfoxide (DMSO) was from Fisher Scientific. AMPA was from Cayman Chemical and NBQX was from Alomone Labs. Recombinant GluA1 and 14-3-3ε were from Origene. Recombinant Nedd4-2 was from Abnova. R18 was from Sigma-Aldrich. Cycloheximide, poly-D-lysine and Protein A/G beads were from Santa Cruz Biotechnology. The antibodies used in this study were purchased from Santa Cruz Biotechnology (anti-α-Tubulin), Cell Signaling (anti-Nedd4-2, anti-pan-14-3-3, anti-N-cadherin and anti-Ubiquitin), Millipore (anti-GluA1), Abcam (anti-MAP2), Thermo Scientific (anti-HA) and GenScript Corporation (anti-Gapdh). The epilepsy-associated mutations were generated using site-directed mutagenesis reagent (Agilent) to introduce mutations into pCI-HA-Nedd4-2 [15 (link)]. The primers used are as below.
S233L: 5’-GGACGTGTCCTCGGAGTTGGACAATAACATCAGAC-3’,
5’-GTCTGATGTTATTGTCCAACTCCGAGGACACGTCC-3’;
E271A: 5’- GGGCGGGGATGTCCCCGCGCCTTGGGAGACCATTTC-3’,
5’- GAAATGGTCTCCCAAGGCGCGGGGACATCCCCGCCC-3’;
H515P: 5’- CGTTTGAAATTTCCAGTACCTATGCGGTCAAAGACATC-3’,
5’- GATGTCTTTGACCGCATAGGTACTGGAAATTTCAAACG-3’.

Total proteins were extracted from cultured cells or homogenized renal tissues using radioimmunoprecipitation assay (RIPA) buffer (Sigma), and western blotting analysis was performed as described previously18 (link). The following primary antibodies were used: rabbit polyclonal anti-Sirt1 (1:800; Sigma), anti-cleaved caspase-3 (1:1000; Cell Signaling Technology, Danvers, MA), anti-nitrotyrosine (1:1000; Santa Cruz), anti-AT1R (1:500; Millipore), anti-AT2R (1:500; Millipore), anti-NF-κB (p65) (1:5000; Enzo Life Science, Farmingdale, NY, USA), anti-acetyl (K310)-NF-kB p65 (1:1000; Abcam, Cambridge, MA), anti-phospho (Ser32)-IκBα (1:1000; Cell Signaling), anti-connective tissue growth factor (CTGF; 1:1000; Peprotech, Rocky Hill, NJ, USA), hamster monoclonal anti-monocyte chemotactic protein 1 (MCP-1; 1:500; Abcam), mouse monoclonal anti-fibronectin (1:400; Abcam), anti-GAPDH (1:2000; GenScript, Piscataway, NJ, USA), and anti-proliferating cell nuclear antigen (PCNA, 1:1000; GeneTex Inc., Irvine, CA). The secondary horseradish peroxidase (HRP)-conjugated antibodies included goat anti-rabbit IgG (Santa Cruz Biotechnology), goat anti-mouse IgG (PerkinElmer, Waltham, MA, USA), and goat anti-hamster IgG (Invitrogen) diluted 1:5000. Specific signals were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore).

Total proteins were extracted with Laemmli sample buffer, boiled, and resolved in 10% SDS-PAGE gel, transferred onto polyvinylidene fluoride (PVDF) membrane, blocked with nonfat dry milk, and probed with indicated antibodies, respectively. Anti-GAPDH (#A01622, GenScript), anti-AAV2 Rep (clone 303.9, #03-65169, American Research Products), anti-AAV2 VP (clone A69, #03-61057, American Research Products), and anti-c-Myc (clone 9E10, #SC-40, Santa Cruz) were purchased; anti-HBoV1 NS and anti-HBoV1 NP1 were homemade and have been described previously.⁵³ (link)

Cells were starved and pretreated with U0126 (5 μM), SB203580 (5 μM), H89 (10 μM), SP600125 (5 μM) or wortmannin (0.5 μM) for 1 h and then stimulated with 10 nM TSH for 60 min. Cells were lysed in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA and 1% Triton-X 100) with a protease inhibitor cocktail for 30 min at 4°C. Lysates were quantified using a BCA protein assay kit (Pierce) according to the manufacturer's instructions. Proteins in the lysate were separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore). After blocking with 5% skimmed milk, the membranes were incubated with anti-PAK4 (1:1000), anti-p-PAK4 (1:1000), anti-PKA Cα(1:1000) (Cell Signaling Technology, Beverly, MA, USA), anti-TSHR (1:1000) (Santa Cruz), cAMP (1:1000) (Sigma) and anti-GAPDH (1:2000) (GenScript Corporation, Nanjing, China) antibodies. U0126, SB203580, H89, SP600125, wortmannin, and forskolin were purchased from BioTime Technology.