K48 ubiquitin

The following antibodies were used: USP14 [9 (link)]; β-tubulin (Developmental Studies Hybridoma Bank, Iowa City, IA); Rpt1 and MLK3 (Santa Cruz Biotechnologies, Dallas, TX); Ubiquitin (UAB Hybridoma Facility, Birmingham, AL), K48 Ubiquitin and K63 Ubiquitin (Millipore, Billerica, MA); pMKK4, MKK4, pJNK, and JNK (Cell Signaling Technology, Danvers, Massachusetts).

FLAG-tagged Bcl-3, or HA-tagged Smad3 were cloned into pcDNA3.1 and pcDNA3.0.
All PCR products were confirmed by sequencing. The following antibodies were
used: anti-Bcl-3, c-Myc, cyclinD1, p27, p21, vimentin, E-cadherin,
N-cadherin, ID1, FLAG, HA, ubiquitin (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA, USA), anti-Snail, ID3, RBX1 (Proteintech), anti-phos-ERK,
phos-AKT, AKT, phos-Smad3, Smad3, phos-Smad2, Smad2, Lamin A/C (Cell
Signaling Technology, Boston, MA, USA), anti-GAPDH, donkey anti-Goat IgG
(HRP), goat anti-Mouse IgG (HRP), goat anti-Rabbit IgG (HRP) (KANGCHEN,
Shanghai, China), anti-Actin (Sigma-Aldrich, St. Louis, MO, USA),
anti-K63-ubiquitin, K48-ubiquitin (Millipore, Billerica, MA, USA), donkey
anti-goat coupled to AlexaFluor®488, and donkey anti-mouse or rabbit IgG
coupled to AlexaFluor680 (Invitrogen, Carlsbad, CA, USA). Anti-Smad3
Ser423/425 (Cell Signaling Technology), anti-Smad2/3 pT8,
anti-Smad2/3 pT179, anti-Smad3 pS204, anti-Smad3 pS208, anti-Smad3 pS213
were kind gifts from Dr Liu Fang.

Protein concentration was determined using the Lowry method with bovine serum albumin as a standard (Lowry et al., 1951 (link)). Primary hippocampal neurons or hippocampal slices were solubilized on ice with RIPA buffer (1% Triton X-100, 1% sodiumdeoxycholate, 0.1% SDS, 0.15MNaCl, 0.01Msodium phosphate, pH7.2) followed by sonication. Immunoblotting was performed after transferring SDS-PAGE gels to nitrocellulose membrane and blocking with 5% low-fat milk for 1 hr at room temperature. The proteins of interest were visualized after incubation with primary antibodies (α-synuclein 1:1000 BD Biosciences (San Jose, CA) #610787; Synapsin-1 1:1000 Synaptic System (Goettingen, Germany) #106001; Munc18–1 1:1000 Synaptic System #116 002; Arp2/3 1:1000 Novus Biologicals (Centennial, CO) # NBP188852; Ubiquitin K48 1:1000 EMD Millipore (Burlington, MA) #05–1307) by chemiluminescence using peroxidase-conjugated secondary antibodies in LAS-3000 Imaging System (Fujifilm, Tokyo, Japan). Densitometric quantification of the immunoblotted membranes was performed using ImageJ (NIH). All protein quantifications were done upon normalization of protein levels to Ponceau staining. Ponceau normalization was chosen over comparison to actin as our work and others showed that CB₁-iLTD induces modification of actin cytoskeleton.