Rabbit anti myc tag

Total protein from N2a cells was harvested in PBS supplemented with 1% Triton-X 100. Samples were denatured 30 min at room temperature following addition of standard Laemmli SDS-PAGE loading buffer to limit aggregate formation [47 (link), 48 ]. Proteins were separated on 10% polyacrylamide denaturing gels and transferred to nitrocellulose membranes. Blocking and antibody dilution was carried out in PBS containing 2% BSA and 0.05% Tween.
Western blots were probed with mouse anti-Cx32 clone 7c6.7c (Elliot Hertzberg) 1:1000, rabbit anti-Myc tag (AbCam) 1:2000, rabbit anti-acetylated proteins (Ack, Cell Signaling Technologies) 1:1000, mouse anti-ubiquitinated proteins clone P4G7 (Covance)1:200, and mouse anti-β-tubulin clone 3F3-G2 at 1:2000. Goat anti-rabbit IRdye800 and goat anti-mouse IRdye700 secondary antibodies (LiCor) were used for detection and digital images were obtained with the Odyssey imaging system (LiCor).
Western blots were also analyzed using GelQuant.NET software (http://biochemlabsolutions.com/GelQuantNET.html), normalizing to actin or β-tubulin signal, where appropriate. Fold change was calculated relative to untreated WT signals for each probe.

Samples were collected in 2% SDS in RIPA buffer containing protease and phosphatase inhibitor cocktail. Lysates were briefly sonicated on ice and centrifuged at 13 200 rpm for 10 min in a tabletop Eppendorf 5414 centrifuge. Proteins were resolved by standard electrophoresis conditions on 10–12% polyacrylamide precast mini-Protean TGX gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were rinsed in Tris-buffered saline (TBS-T, 0.1% Tween 20) for 5 min at room temperature and subsequently blocked in 5% weight/volume nonfat milk in TBS-T for 1 h at room temperature. Samples were incubated for 1 h at room temperature or overnight at 4°C with the following antibodies: rabbit anti-SLC7A5 (1:1 000; Cell Signaling Technology; #5347), rabbit anti-Myc-tag (1:1 000; Cell Signaling Technology; 71D10, #2278), and rabbit anti-Akt (pan) (1:1 000; Cell Signaling Technology; 11E7, #4685). Following an additional five rinses each of 10 min in TBS-T, samples were incubated for 1 h at room temperature with donkey or goat anti-rabbit antibodies in blocking buffer and then subjected to four 15-min washes in TBS-T and visualized using Bio-Rad Chemidoc MP imaging system. PVDF membranes were stripped for 5–15 min at room temperature using Restore Western Blot Stripping Buffer (#21059, Thermo Fisher Scientific) before probing for loading controls.

Primary antibodies used for Western blots were as follows: rabbit anti-Myc tag (Cell Signaling, 71D10), mouse anti-β-actin (Sigma Aldrich, A5441), mouse anti-GAPDH (ABCAM, ab8245), rabbit anti-PDI (EnzoLife, SPA890), mouse anti-PDI (Thermo, RL90- MA3019), mouse anti-RhoGDIα (ABCAM, ab135252), mouse anti-RhoGDIα (Santa Cruz, B-10, sc-13120), rabbit anti-RhoGDIα (ABCAM, ab53850), rabbit anti-RhoGDIα (Santa Cruz, A-20 sc360), goat anti-GST (ABCAM, ab6613). Secondary antibodies were fluorescent antibodies from LI-COR.

Protein concentrations from total lysate supernatants were calculated (Pierce 660 nm protein assay reagent; Thermo Scientific, catalog no.: 22660), and samples were diluted to the same concentration in LDS sample buffer (National Diagnostics; catalog no.: EC-887) and heated in a 37 °C water bath for 15 min. Total lysate (5–10 μg total protein; typically one tenth of the 15% total lysate supernatant fraction) and IP samples (20 μl; 40% of eluent) were subject to SDS polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and incubated with 5% dry nonfat milk (Boston Bioproducts; catalog no.: P-1400) in PBS containing 0.1% Tween-20 (PBST). Membranes were then incubated with primary antibody in 1% milk in PBST as indicated in the text: mouse anti-GFP (Roche; catalog no.: 11814460001), rabbit anti-myc tag (Cell Signaling Technology; catalog no.: 2278), and rabbit anti-mCherry (ProteinTech; catalog no.: 26765-1-AP). Followed by washing in PBST, membranes were incubated with secondary antibodies conjugated to horseradish peroxidase in 1% milk in PBST: mouse anti-rabbit (Jackson ImmunoResearch; catalog no.: 211-032-171) and goat antimouse (Jackson ImmunoResearch; catalog no.: 115-035-174). Protein bands were visualized digitally following application of ECL reagent with the KwikQuant Imager (Kindle Biosciences; catalog no.: D1001).

Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline with 0.1% Triton X-100 for 30 min. A standard staining protocol was used. Primary antibodies were used as indicated: Chicken anti-GFP (1:1000, Abcam ab13970), Mouse anti-Lamin (1:500, DSHB #ADL84.12), Mouse anti-Calnexin99A (1:5, DSHB #Cnx99A 6-2-1), Mouse anti-ATP5A (1:100, Abcam ab14748), Rabbit anti-Arl8 (1:500, DSHB Arl8), Goat anti-GMAP (1:2000, DSHB, #GMAP), Goat anti-Golgin245 (1:2000, DSHB, #Golgin245), Rabbit anti-Ref2P (1:500, Abcam #ab178440), Rabbit anti-Myc-tag (1:1000, Cell Signaling Technology #2278S). Secondary antibodies with Alexa Fluor conjugates and DAPI (Molecular Probes #D-1306) were used at 1:1000. Images were obtained using a GE IN Cell 6000 automated confocal microscope with a 60x objective. Time-lapse videos were generated by imaging every 30 s over a 2 hr period.

Tris, NaCl, and SDS for molecular biology and buffer preparation were purchased from Sigma‐Aldrich. Cell culture media and supplements were from Invitrogen (La Jolla, CA, USA). Antibodies were purchased from commercial sources as follows: anti‐EGFR (IHC‐00005; Bethyl Laboratories, Montigny, TX, USA), anti‐phospho‐EGFR [Tyr1068, Tyr992, and Tyr845 (Cell Signaling, Danvers, MA, USA)], anti‐TRAF4 (sc‐1920; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti‐HA (H9658; Sigma‐Aldrich), Rabbit anti‐HA (Cell Signaling), mouse anti‐Omni and rabbit anti‐Omni (Santa Cruz Biotechnology), rabbit anti‐myc tag (Cell Signaling), mouse anti‐V5 antibody (Invitrogen), anti‐phospho‐ERK5 (Cell Signaling), anti‐ERK5 (Cell Signaling), anti‐phospho‐ERK1,2 (Santa Cruz Biotechnology), anti‐ERK1,2 (Cell Signaling), anti‐AKT (Cell Signaling), anti‐phospho‐AKT (Ser473; Cell Signaling), anti‐β‐actin (Cell Signaling). Recombinant human EGF was purchased from R&D (Minneapolis, MN, USA). Doxycycline and DMSO were purchased from Sigma‐Aldrich.