GBCBIO公司的RIPA Lysis Buffer登上cells
2021/8/26 15:33:09 點擊：
Abstract
RhoA-GTPase (RhoA) is widely regarded as a key molecular switch to inhibit neurite outgrowth by rigidifying the actin cytoskeleton. However, during neurite outgrowth, whether and how microtubule dynamics are regulated by RhoA remains to be elucidated. Herein, CT04 and Y27632 were used to inactivate RhoA and its downstream effector Rho-associated coiled coil-forming kinase (ROCK), while the RhoAQ63L lentiviral vector was utilized to overexpress the constitutively activated RhoA in dorsal root ganglion (DRG) neurons or neuronal differentiated PC12 cells. The current data illustrate that the RhoA signaling pathway negatively modulates neurite outgrowth and elevates the expression of Glu-tubulin (a marker for a stabilized microtubule). Meanwhile, the microtubule-severing proteins spastin and p60-katanin were downregulated by the RhoA signaling pathway. When spastin and p60-katanin were knocked down, the effects of RhoA inhibition on neurite outgrowth were significantly reversed. Taken together, this study demonstrates that the RhoA pathway-mediated inhibition of neurite outgrowth is not only related to the modulation of microfilament dynamics but is also attributable to the regulation of the expression of spastin and p60-katanin and thus influences microtubule dynamics.
Keywords: neurite outgrowth; RhoA signaling pathway; spastin; p60-katanin; microtubule-severing proteins; Glu-tubulin
2.4. Immunocytochemistry
At the designated time points, the subjected cells were fixed with 4% (w/v) paraformaldehyde (PFA) for 20 min and washed three times in 0.01 M PBS. This was followed by permeabilization with 0.5% Triton X-100 (Sigma, St. Louis, MO, USA) for 30 min. Cells were then blocked with 5% bovine serum albumin (BSA, GBCBIO Technologies, Guangzhou, China) in 0.01 M PBS for 1 h, followed by incubation with primary antibodies diluted in 1% BSA overnight at 4 °C. Alexa 488 and/or 568 fluorescent-conjugated secondary antibodies (1:400, Life Technologies, Gaithersburg, MD, USA) were applied for 2 h at room temperature, and the nuclei of all cells were counterstained by 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI, Sigma, St. Louis, MO, USA) for 2 min. The primary antibodies used for immunocytochemistry were as follows: mouse anti-β-tubulin III (1:400, Sigma, St. Louis, MO, USA), mouse anti-spastin (1:200, Sigma, St. Louis, MO, USA), and rabbit anti-p60-katanin (1:200, Proteintech, Chicago, IL, USA). After DAPI counterstaining, the cells were mounted with an anti-fading mounting medium (Vector, Burlingame, CA, USA), and images were captured by a fluorescent microscope (Leica, Wetzlar, Germany). Five images with high-power fields at 400x magnification per well were analyzed, and all images used for the quantification of fluorescence intensities were acquired and generated using identical imaging parameters (e.g., laser power, exposure, pixel saturation, gain, etc.). The fluorescence intensities of spastin and p60-katanin in each cell were assessed using the Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). Finally, the obtained single-channel fluorescent images were merged using the Photoshop 6.0 software (Adobe, San Jose, CA, USA).
2.6. Western Blotting
For Western blotting, subjected cells were washed twice with ice-cold PBS, scraped, and lysed in a RIPA buffer (GBCBIO Technologies, Guangzhou, China) containing a protease inhibitor cocktail (1:100, Cell Signaling Technology, Danvers, MA, USA ). Lysates were incubated on ice for 30 min and centrifuged (14,462 × g, 20 min, 4 °C) in order to collect the supernatant. Extracts were separated in an SDS-PAGE sample buffer on 10% SDS-PAGE gels and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). Blots were blocked with 5% BSA for 1 h and incubated with primary antibodies overnight at 4 °C and then incubated with HRP-conjugated secondary antibodies (Promega, Madison, WI, USA) for 2 h at room temperature. The following primary antibodies were used: rabbit anti-GAPDH (1:3000, Multi Sciences, Hangzhou, China), mouse anti-β-actin (1:2000, Multi Sciences, Hangzhou, China), mouse anti-α-tubulin (1:2000, Abcam, Cambridge, UK), rabbit anti-detyrosinated alpha tubulin (Glu-tubulin, 1:500, Abcam, Cambridge, UK), mouse anti-spastin (1:1000, Sigma, St. Louis, MO, USA), rabbit anti-p60-katanin (1:500, Proteintech, Chicago, IL, USA), rabbit anti-phospho-p38 (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-p38 (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-PTEN (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-PI3K (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-AKT (Ser473) (1:1000, Cell Signaling Technology, Danvers, MA, USA), and rabbit anti-AKT (1:1000, Cell Signaling Technology, Danvers, MA, USA). The Western blotting for each protein was repeated three times for each test. Immunoreactive protein bands were detected and imaged by enhanced chemiluminescence (ECL, Millipore, Burlington, MA, USA) using a Lumazone system (Roper, Trenton, NJ, USA). The integrated optical density (IOD) of each lane was quantified with the Image-Pro Plus software, and the expression levels of the targeted proteins were calculated by dividing the IOD of the targeted proteins by the IOD of the GAPDH or β-actin.
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