Computational protocol: BBS4 Is Necessary for Ciliary Localization of TrkB Receptor and Activation by BDNF

Similar protocols

Protocol publication

[…] Cells were washed in ice-cold PBS and harvested in ice-cold buffer containing 50 mM Tris, 150 mM MgCl2, 1% NP-40, protease inhibitor (Sigma) and phosphatase inhibitor (Sigma). Cells were incubated in lysis buffer on ice for 15 minutes, vortexing every 5 minutes. Lysates were centrifuged in an Eppendorf 5415R at 4°C, 8000xg for 10 minutes. Supernatant was collected and incubated 1∶1 in Laemmli sample buffer (BioRad) plus β-mercaptoethanol for 10minutes and boiled for 5 minutes. Samples were run on a NuPage 4–12% Bis-Tris gel (Life Technologies) with MOPS running buffer (Life Technologies). Proteins were transferred onto a nitrocellulose membrane at 30 V for 90 minutes on ice. Membranes were blocked in 5% milk in TBST for one hour at room temperature with rocking. Membranes were incubated overnight in primary antibody, washed the following day in TBST and incubated in the appropriate secondary antibody. Blots were developed using ECL substrate (Pierce) using a FluorChemQ system and AlphaView Software. Blots were probed for detection of a TRKB band at approximately 95 kDa MW and subsequently stripped for detection of phosphorylated TRKB (pTRKB), also near 95 kDa, followed by stripping for Actin loading control (42 kDa) using ReStore Western Blot Stripping Buffer (Thermo Scientific). The following primary antibodies and concentrations were used: Anti-TRKB (BD Transduction Laboratories 610101, 1∶1000), Anti-phosphoNTRK2/pTRKB (Y515; Sigma-Aldrich SAB4503785, 1∶1000 or Y705; Abcam ab52191, 1∶1000), Anti-ACTIN (Sigma-Aldrich A2103, 1∶1500). The following secondary antibodies and concentrations were used: Anti-mouse IgG HRP-linked (Cell Signaling Technology 7076, 1∶2500), Anti-rabbit IgG HRP-linked (Cell Signaling Technology 7074, 1∶2500). Western blots were quantified by densitometry analysis using ImageJ software and quantification of each band relative to ACTIN. TRKB activation was measured as the ratio of pTRKB protein to TRKB protein for each lane. Average activation was calculated from a minimum of 3 (and up to 5) separate experiments. [...] Cells plated on coverslips and transfected were fixed at 100% confluency in ice cold 1∶1 methanol:acetone for 2 minutes followed by two 1XPBS washes. Double immunostaining was carried out starting with a one-hour PBS/10% serum/1% BSA block, one-hour primary antibody incubation at room temperature, second primary antibody incubation for one-hour at room temperature, and a one-hour secondary antibody incubation, with both secondary antibodies mixed together. The following primary antibodies were used: anti-TRKB (BD Transduction Laboratories 610101 1∶1000), anti-pTRKB (Abcam ab52191, 1∶1000), anti-ARL13B (Proteintech 17711-1-AP, 1∶1000), anti-γ-tubulin (Sigma-Aldrich T5192, 1∶1000. Species-specific secondary antibodies (AlexaFluor, Life Technologies) were used at 1∶1000. Following primary and secondary antibody incubations, coverslips were washed in 1XPBS, incubated in DAPI (0.2 µg/ml in PBS), mounted in Prolong Gold Antifade (Life Technologies), and imaged at 100× magnification with an Olympus IX50 with cellSens imaging software using deconvolution. Co-localization was quantified by assessing overlap of fluorescence in a single focal plane between ciliary markers and TRKB or pTRKB and counting the proportion of either basal bodies or axonemes that co-localize in 75–100 cells across a minimum of 3 separate experiments. Measurement of cilia length was calculated using ImageJ software on imaged hTERT-RPE1 cells labeled with antibodies against ARL13B. […]

Pipeline specifications

Software tools ImageJ, cellSens
Application Microscopic phenotype analysis
Diseases Neural Tube Defects, Bardet-Biedl Syndrome