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1

Synaptophysin and VGLUT1 Quantification

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Total cells were lysed in 2% SDS, briefly sonicated and then incubated at 37 °C for 30 min with 1% 2-mercanptoethanol. After SDS-PAGE, samples were transferred to polyvinylidene difluoride membrane. The membranes were incubated in 5% Slim milk and 0.2% Tween-20, treated with primary antibodies and then probed with HRP-conjugated secondary antibodies (GE Healthcare) and chemiluminescent signals were acquired. Band intensities were measured using MultiGauge software (GE Healthcare) or ImageJ software (NIH). The ratio of synaptophysin 1 or VGLUT1 to β-III-tubulin were acquired and normalized to the value of control.

Hayashi Y., Nishimune H., Hozumi K., Saga Y., Harada A., Yuzaki M., Iwatsubo T., Kopan R, & Tomita T. (2016). A novel non-canonical Notch signaling regulates expression of synaptic vesicle proteins in excitatory neurons. Scientific Reports, 6, 23969.

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2

Western Blotting of Cell Lysates

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Western blotting of cell lysis was performed using the experimental procedures as previously described [8 (link), 28 (link), 35 (link)–37 (link)]. Antibodies used were anti-nestin (1:1000, Fisher Invitrogen #PIPA511887/L/N SH2420723H and Cell Signaling #10959s/L/N 1), anti-GAPDH (1: 1000, Santa Cruz Biotechnology #sc-32233/L/N K0315), anti phospho-myosin light chain (Ser-19, Santa Cruz Biotechnology, 1:500), anti-myosin light chain (1:1000, custom made) [8 (link), 38 (link), 39 (link)], anti-vimentin (BD Biosciences, #550515, L/N 3214517, 1:10,000), anti-phospho-vimentin (Ser-56) (custom made) [8 (link), 40 (link), 41 (link)], anti-Plk1 (1:1000, EMD Millipore, #05-844, L/N 2477015), anti-phospho-Plk1 (T210) (Cell Signaling 9062 S1), and anti-phospho-nestin (Thr-315) (Santa Cruz Biotechnology, 1:500, sc-377538, J2820).
The antibodies were validated by examining the molecular weight of target proteins. In addition, anti-nestin and anti-Plk1 were validated by using KD cells. Finally, vendors have provided datasheet to show that antibodies were validated by positive controls. The levels of proteins were quantified by scanning densitometry of immunoblots (Fuji Multi Gauge Software or GE IQTL software). The luminescent signals from all immunoblots were within the linear range.

Wang Y., Liao G., Wu Y., Wang R, & Tang D.D. (2023). The intermediate filament protein nestin serves as a molecular hub for smooth muscle cytoskeletal signaling. Respiratory Research, 24, 157.

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3

Quantifying Brain MAO Enzyme Activity

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MAO activity was determined as previously described [12 (link)]. Briefly, brain regions were freeze fractured and homogenized in reaction buffer containing 50 mM NaCl (Fisher Scientific, Fair Lawn, NJ), 10% glycerol, 1 mM EDTA, 50 mM HEPES, pH 7.4 (USB Corporation, Cleveland, OH). Lysates were clarified at 16,000 × g for 15 min at 4 °C and the supernatant protein concentrations determined using the DC Protein Assay (Bio-Rad, Hercules, CA) as above. Sample protein concentrations were normalized within regions with reaction buffer. MAO activity was tested using the MAO-Glo Assay System (Promega, Madison, WI) and quantified with an ImageQuant LAS-4000 CCD camera quantitative imager (GE Healthcare, Piscataway, NJ) coupled to Multi Gauge Software (GE Healthcare, Piscataway, NJ).

Moon M.L., Joesting J.J., Lawson M.A., Chiu G.S., Blevins N.A., Kwakwa K.A, & Freund G.G. (2014). The saturated fatty acid, palmitic acid, induces anxiety-like behavior in mice. Metabolism: clinical and experimental, 63(9), 1131-1140.

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4

Western Blotting and Co-Immunoprecipitation Protocols

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Western blotting of cell lysis and co‐immunoprecipitation were performed using the experimental procedures as previously described.⁸, ²⁶, ³⁰, ³², ³³ Antibodies used were anti‐Abi1 (1:1500, Sigma #A5106‐200UL, L/N 076M4842V), anti‐GAPDH (1:1500, Ambion #AM4300, L/N 1311029), and anti‐α‐actin (Sigma, A2647, #22190320, 1:3000), anti‐phospho‐myosin light chain (Ser‐19, Santa Cruz Biotechnology, 1:500), Ac‐lysine Antibody (Santa Cruz Biotechnology, S7F8, sc‐81623), anti‐N‐WASP (Santa Cruz Biotechnology, #sc‐10121, L/N G2211 1:500), anti‐myosin light chain (1:1000, a gift of Dr Gunst), anti‐phospho‐N‐WASP (Y256) (EMD Millipore, #AB1966, L/N 2795491,2838736, 1:1000), and anti‐vimentin (BD Biosciences, #550515, L/N 3214517, 1:10 000). Phospho‐vimentin (Ser56) antibody (1:500) was produced as previously described.²⁹, ³⁴ The antibodies were validated by examining the molecular weight of target proteins. In addition, anti‐Abi1 was validated using Abi1 KD cells.¹⁷ Finally, vendors have provided datasheet to show that antibodies were validated by positive controls. The levels of proteins were quantified by scanning densitometry of immunoblots (Fuji Multi Gauge Software or GE IQTL software). The luminescent signals from all immunoblots were within the linear range.

Wang Y., Liao G., Wang R, & Tang D.D. (2021). Acetylation of Abelson interactor 1 at K416 regulates actin cytoskeleton and smooth muscle contraction. The FASEB Journal, 35(9), e21811.

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5

Separation of Nuclear Proteins by SDS-PAGE

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For separating nuclear-enriched protein extracts by SDS-PAGE, equal volumes of nuclear-enriched extracts were loaded. To separate total protein extracts, equal amounts of protein were resolved by SDS-PAGE. Protein samples were subsequently blotted onto PVDF membranes. After blotting, membranes were blocked with Rotiblock (Roth) reagent and incubated with the respective primary antibody followed by a horseradish peroxidase (HRP)-conjugated secondary antibody. HRP activity was detected using the SuperSignal West Femto Maximum Sensitivity kit (Thermo Scientific) and visualized by a LAS-4000 Mini bioimager (GE Healthcare Life Sciences). Signal intensities were quantified using Multi-Gauge software (GE Healthcare Life Sciences). Commercial antibodies used were HRP-conjugated α-HA (Roche), α-Histone H3 (Abcam), α-HSC70 (Stressgen), α-α-Tubulin (Sigma-Aldrich), α-rabbit IgG-HRP (Sigma-Aldrich) and α-mouse IgG-HRP (Sigma-Aldrich). α-SPA2 and α-COP1 antibodies were described previously in [42 (link)]. α-cry1 [67 ] and α-cry2 [68 (link)] antibodies were used to detect cry1 and cry2, respectively.

Chen S., Lory N., Stauber J, & Hoecker U. (2015). Photoreceptor Specificity in the Light-Induced and COP1-Mediated Rapid Degradation of the Repressor of Photomorphogenesis SPA2 in Arabidopsis. PLoS Genetics, 11(9), e1005516.

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6

Western Blot Analysis of Protein Signaling

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Tissues were lysed in NP-40 buffer supplemented with protease and phosphatase inhibitor cocktails (catalogue nos. 04080-11 and 07574-61; Nacalai Tesque, Kyoto, Japan). Total protein concentrations were determined by using the Pierce 660 nm Protein Assay Reagent (Thermo Fisher Scientific, Rockford, MA, USA). Proteins were separated by electrophoresis through a 4–15% sodium dodecyl sulphate–polyacrylamide gel (Bio-Rad, Hercules, CA, USA) and were transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was incubated in blocking reagent (Toyobo, Osaka, Japan) at room temperature for 20 min and then was incubated at 4 °C overnight with the appropriate primary antibody diluted in Can Get Signal Solution 1 (Toyobo). All primary antibodies were from Cell Signaling Technology (Danvers, MA, USA): anti-phospho-Akt (Ser⁴⁷³) (catalogue no. 9271), anti-Akt (catalogue no. 9272), anti-ACC (catalogue no. 3662), anti-FASN (catalogue no. 3180), and anti-GAPDH (catalogue no. 2118). An image of the membrane was obtained by using a LAS-4000 mini luminescent image analyser (GE Healthcare UK, Little Chalfont, England, UK), and band intensities were quantitated by using Multi Gauge software (version 3.11, GE Healthcare UK).

Bae C.R., Hino J., Hosoda H., Arai Y., Son C., Makino H., Tokudome T., Tomita T., Kimura T., Nojiri T., Hosoda K., Miyazato M, & Kangawa K. (2017). Overexpression of C-type Natriuretic Peptide in Endothelial Cells Protects against Insulin Resistance and Inflammation during Diet-induced Obesity. Scientific Reports, 7, 9807.

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7

Quantification of Neurotransmitter Receptors

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We used 5-month-old naïve OM and S5B rats. Slides with mounted brain sections at the level of dorsal striatum that were stored at −80°C were gradually brought back to room temperature and then exposed to receptor autoradiography procedures using [³H]spiperone, [³H]SCH23390, [³H]MPEP, and [³H]SR141716A as previously described^{21 (link), 26 (link)–28 (link)}. Radioactivity concentrations were verified via liquid scintillation counting. For [³H]spiperone and [³H]SCH23390 experiments, slides were placed in BAS-TR 2025 (GE Healthcare, Piscataway, NJ) phosphor imaging plates for 7 days along with [¹⁴C] standards (GE Healthcare, Piscataway, NJ) (calibrated against ³H-impregnated brain paste standards). Imaging plates were developed using an FLA-7000 phosphorimager (GE Healthcare, Piscataway NJ). Using Multigauge® software (GE Healthcare, Piscataway, NJ), regions of interest (ROIs) were drawn on the dorsolateral (DLST) and dorsomedial striatum (DMST) of each section. Values were averaged and initially expressed as PSL/mm² and subsequently converted to dpm/mg with the use of [¹⁴C] standards. For [³H]MPEP and [³H]SR141716A experiments, slides were imaged using a β-imager (Biospace Lab, France). Scanning and image analysis methods were performed as previously described^{21 (link)}.

Michaelides M., Miller M.L., Egervari G., Primeaux S.D., Gomez J.L., Ellis R.J., Landry J.A., Szutorisz H., Hoffman A.F., Lupica C.R., Loos R.J., Thanos P.K., Bray G.A., Neumaier J.F., Zachariou V., Wang G.J., Volkow N.D, & Hurd Y.L. (2018). Striatal Rgs4 regulates feeding and susceptibility to diet-induced obesity. Molecular psychiatry, 25(9), 2058-2069.

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8

Western Blotting and Coimmunoprecipitation Protocols

Cited 3 times

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Western blotting of cell lysis and coimmunoprecipitation were performed using the experimental procedures as previously described (8 (link), 26 (link), 30 (link), 32 (link), 33 (link)). Antibodies used were anti-Abi1 (1:1500, Sigma #A5106–200UL, L/N 076M4842V), anti-GAPDH (1:1500, Ambion #AM4300, L/N 1311029) and anti α-actin (Sigma, A2647, #22190320, 1:3000), Anti phospho-myosin light chain (Ser-19, Santa Cruz Biotechnology, 1:500), Ac-lysine Antibody (Santa Cruz Biotechnology, S7F8, sc-81623), anti-N-WASP (Santa Cruz Biotechnology, #sc-10121, L/N G2211 1:500), anti-myosin light chain (1:1000, a gift of Dr. Gunst), anti-phospho-N-WASP (Y256) (EMD Millipore, #AB1966, L/N 2795491,2838736, 1:1000), anti-vimentin (BD Biosciences, #550515, L/N 3214517, 1:10000). Phospho-vimentin (Ser56) antibody (1:500) was produced as previously described (29 , 34 ). The antibodies were validated by examining the molecular weight of target proteins. In addition, anti-Abi1 was validated by using Abi1 KD cells (17 (link)). Finally, vendors have provided datasheet to show that antibodies were validated by positive controls. The levels of proteins were quantified by scanning densitometry of immunoblots (Fuji Multi Gauge Software or GE IQTL software). The luminescent signals from all immunoblots were within the linear range.

Wang Y., Liao G., Wang R, & Tang D.D. (2021). Acetylation of Abelson interactor 1 at K416 regulates actin cytoskeleton and smooth muscle contraction. The FASEB Journal, 35(9), e21811.

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9

Quantification of Neurotransmitter Receptors

Cited 1 time

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We used 5-month-old naïve OM and S5B rats. Slides with mounted brain sections at the level of dorsal striatum that were stored at −80°C were gradually brought back to room temperature and then exposed to receptor autoradiography procedures using [³H]spiperone, [³H]SCH23390, [³H]MPEP, and [³H]SR141716A as previously described^{21 (link), 26 (link)–28 (link)}. Radioactivity concentrations were verified via liquid scintillation counting. For [³H]spiperone and [³H]SCH23390 experiments, slides were placed in BAS-TR 2025 (GE Healthcare, Piscataway, NJ) phosphor imaging plates for 7 days along with [¹⁴C] standards (GE Healthcare, Piscataway, NJ) (calibrated against ³H-impregnated brain paste standards). Imaging plates were developed using an FLA-7000 phosphorimager (GE Healthcare, Piscataway NJ). Using Multigauge® software (GE Healthcare, Piscataway, NJ), regions of interest (ROIs) were drawn on the dorsolateral (DLST) and dorsomedial striatum (DMST) of each section. Values were averaged and initially expressed as PSL/mm² and subsequently converted to dpm/mg with the use of [¹⁴C] standards. For [³H]MPEP and [³H]SR141716A experiments, slides were imaged using a β-imager (Biospace Lab, France). Scanning and image analysis methods were performed as previously described^{21 (link)}.

Michaelides M., Miller M.L., Egervari G., Primeaux S.D., Gomez J.L., Ellis R.J., Landry J.A., Szutorisz H., Hoffman A.F., Lupica C.R., Loos R.J., Thanos P.K., Bray G.A., Neumaier J.F., Zachariou V., Wang G.J., Volkow N.D, & Hurd Y.L. (2018). Striatal Rgs4 regulates feeding and susceptibility to diet-induced obesity. Molecular psychiatry, 25(9), 2058-2069.

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10

Western Blot Analysis of Prestin

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The segments of the dissected organs of Corti were collected and homogenized with ultrasonic cell crushers (VCX150, Sonic, Newtown, CT, USA). The supernatants of the homogenates were subjected to 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose blotting membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk in TBS-T (20 mM Tris, 137 mM NaCl, and 0.1% Tween 20, pH 7.6) for 1 h at 27°C, the membranes were incubated with anti-prestin or anti-GAPDH antibodies overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibodies for 1 h at 27°C. The immunobands were detected by a chemiluminescence detection system (GE Healthcare, Chicago, IL, USA) and visualized using the Amersham Imager 600 (AI600, GE Healthcare, Chicago, IL, USA). The intensity of the immunobands was quantified using MultiGauge software (GE Healthcare, Chicago, IL, USA). Data were obtained from four independent experiments.

Chang A., Chen P., Guo S., Xu N., Pan W., Zhang H., Li C, & Tang J. (2018). Specific Influences of Early Acoustic Environments on Cochlear Hair Cells in Postnatal Mice. Neural Plasticity, 2018, 5616930.

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Multi gauge software

Lab products found in correlation

11 protocols using multi gauge software

Synaptophysin and VGLUT1 Quantification

Western Blotting of Cell Lysates

Quantifying Brain MAO Enzyme Activity

Western Blotting and Co-Immunoprecipitation Protocols

Separation of Nuclear Proteins by SDS-PAGE

Western Blot Analysis of Protein Signaling

Quantification of Neurotransmitter Receptors

Western Blotting and Coimmunoprecipitation Protocols

Quantification of Neurotransmitter Receptors

Western Blot Analysis of Prestin

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