The largest database of trusted experimental protocols

4 protocols using anti β arrestin2

1

Immunofluorescence Analysis of Hippocampal Synaptic Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
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.
+ Open protocol
+ Expand
2

Evaluating Apoptosis Signaling Pathways

Check if the same lab product or an alternative is used in the 5 most similar protocols
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).
+ Open protocol
+ Expand
3

Proximity Ligation Assay for Receptor Interactions

Check if the same lab product or an alternative is used in the 5 most similar protocols
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).
+ Open protocol
+ Expand
4

Comprehensive Western Blotting Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
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).
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!