Immunofluorescence experiments were performed according to [73 (link)]. Briefly, hippocampal primary cultures at DIV14 (see above) were fixed in 4% paraformaldehyde (PFA) and 4% sucrose solution for 30 min, followed by permeabilization with phosphate-buffered saline (PBS) at pH 7.4, containing 0.5% Triton X-100, for 3 min. Co-cultures were first blocked for 1 h in PBS containing 1% BSA, 0.2% Triton X-100, and subsequently incubated overnight at 4 °C with primary antibodies in PBS containing 1% BSA and 0.2% Triton X-100. The following antibodies were used: anti-GFP (AbCam Cambridge, UK Ab290), anti-PSD95 (AbCam Cambridge, UK Ab12093), anti-JIP1 (AbCam Cambridge, UK Ab24449), anti-β-arrestin2 (AbCam Cambridge, UK Ab31294), anti-JNK3 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA #PA5-14421). Cells were finally incubated with secondary antibodies (AlexFluor Antibody, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. A concentration of 2 mg/mL Hoechst (Thermo Fisher Scientific, Waltham, MA, USA, 33342) was used to stain nuclei. ProLong Glass Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA) was used as a mounting agent.
Anti β arrestin2
Manufactured by Abcam
Sourced in United Kingdom
Anti-β-arrestin2 is a primary antibody that recognizes the beta-arrestin 2 protein. Beta-arrestin 2 is a cellular regulatory protein involved in the desensitization and internalization of G protein-coupled receptors.
Automatically generated - may contain errors
Lab products found in correlation
Prolong glass antifade mountant, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Anti jnk3, by Thermo Fisher Scientific (1 mentions)
Alexfluor antibody, by Thermo Fisher Scientific (1 mentions)
Anti gfp, by Abcam (1 mentions)
Anti psd95, by Abcam (1 mentions)
Anti jip1, by Abcam (1 mentions)
Anti cleaved parp, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Duolink in situ detection reagents red, by Merck Group (1 mentions)
Anti phospho creb ser133, by Cell Signaling Technology (1 mentions)
Anti cdk2, by Cell Signaling Technology (1 mentions)
Anti cyclin e1, by Cell Signaling Technology (1 mentions)
Anti β arrestin 1, by Abcam (1 mentions)
Anti tubulin, by Beyotime (1 mentions)
Anti p21, by Beyotime (1 mentions)
Anti creb, by Cell Signaling Technology (1 mentions)
Anti p27, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
4 protocols using anti β arrestin2
1
Immunofluorescence Analysis of Hippocampal Synaptic Proteins
2
Evaluating Apoptosis Signaling Pathways
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Cells were harvested and lyzed in RIPA buffer containing protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). The total protein concentration was measured by bicinchoninic acid method. An equal amount of protein from each sample was loaded into 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. After 5% BSA blocking, primary antibodies were incubated at 4°C overnight. Then, the membranes were incubated with secondary antibodies and reacted with enhanced chemiluminescence detection solution. Results were normalized to β-actin. The primary antibodies used were as follows: anti-GPR35 (ab76217), anti-Bcl2 (ab32124), anti-p21 (ab109520), anti-bcl-xl (ab32370), and anti-β-arrestin-2 (ab54790), which were from Abcam. Anticleaved caspase 3 (#9661), antiphosphorylated Akt (#4060), antitotal Akt (#2920), antiphosphorylated S6 (#4858), anti-S6 (#2317), and anticleaved PARP (#5625) were from Cell Signaling Technology (Beverly, MA, USA).
3
Proximity Ligation Assay for Receptor Interactions
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A proximity ligation assay was performed using Duolink in situ Detection Reagents Red (Sigma-Aldrich) according to the manufacturer’s instructions. M1-like HMDMs were probed with primary antibodies (rabbit anti-Rab5a (1:500, Abcam), rabbit anti-C5aR1 (1:250, Abcam), mouse, anti-β-arrestin2 (1:750, Abcam), mouse anti-Gαq/11/14 (1:50), mouse anti-Gαs/o (1:100), mouse anti-Gα12 (1:100), mouse anti-Gα13 (1:50, Santa Cruz Biotechnology), then detected with the supplied secondary antibodies and reagents. M1-like HMDMs were imaged using a confocal microscope (as above) and serial z-sections were acquired at 0.31 μm intervals. The number of proximity ligation fluorescence spots in a single cell was quantified using the “Spots” function, with consistent settings across all images in Imaris v9.2.1 software (Bitplane AG).
4
Comprehensive Western Blotting Analysis
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Western Blotting analysis was performed as described previously 32 (link). Total cell extracts were lysed in cell lysis buffer (Beyotime, P0013), and the lysates were centrifuged at 12,000 rpm for 10 min at 4°C. Samples containing 30 µg protein were loaded on SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 5% nonfat milk or BSA and probed with primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Blots were developed with lumi-light Western blotting substrate detection reagents (Roche, 12015200001). Nuclear- and cytoplasmic-enriched fractions were prepared using Nuclear-Cytosol Extraction Kit (Applygene, P1200), according to manufacturer's instructions. Western Blotting analysis was performed using the anti-p27 (Cell Signaling Technology, 3698S), anti-CREB (Cell Signaling Technology, 9197L), anti-phospho-CREB (Ser133) (Cell Signaling Technology, 9198L), anti-S-phase kinase-associated protein 2 (Skp2) (Cell Signaling Technology, 4313S), anti-CDK2 (Cell Signaling Technology, 2546S), anti-Cyclin E1 (Cell Signaling Technology, 4129S), PARP (Cell Signaling Technology, 9532S), anti-GAPDH (Cell Signaling Technology, 2118L), anti-β-arrestin 1 (Abcam, ab32099), anti-β-arrestin 2 (Abcam, ab54790), anti-tubulin (Beyotime, AT819), anti-cyclin D3 (Beyotime, AC856), anti-p21 (Beyotime, AP021).
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