rabbit anti ppp2r2a  (Proteintech)

Journal: Developmental Biology

Article Title: Rab3Gap1 mediates exocytosis of Claudin-1 and tight junction formation during epidermal barrier acquisition

doi: 10.1016/j.ydbio.2013.04.034

Figure Lengend Snippet: Expression of Ppp2r2a correlates with pAKT activation, cJun dephosphorylation and interaction between Claudin-1 and Occludin during epidermal development. (A) Representative data from immunofluorescent analysis of Ppp2r2a, pSerAkt, and pcJun, and Claudin-1 and Zo-1 of E15.5 and E16.5 embryonic epidermis from 3 different embryos. (B) Representative data from a proximity ligation experiment between Occludin and Claudin-1 and Occludin and Zo-1 antibodies in triplicate embryos. A red dot indicates that the two proteins are within 40 nm of each other, suggestive of interaction. Bar (A and B) 50 µm.

Article Snippet: Primary antibodies and concentrations were as follows: rabbit anti PR55a (Ppp2r2a) (Santa Cruz Biotechnologies, Santa Cruz, US) 1/500; rabbit anti cJun (Cell Signalling Technologies, Danvers, USA) 1/100; rabbit anti pSer63 cJun (Cell Signalling Technologies, Danvers, USA) 1/100; rabbit anti Claudin-1 (Invitrogen, Paisley, UK) 1/500; mouse anti Occludin (Invitrogen, Paisley, UK ) 1/500; Rabbit anti Zonula Occludins-1 (Santa Cruz Biotechnologies, Santa Cruz, US) 1/500; Mouse anti Rab3 (BD biosciences, Oxford, UK) 1/100.

Techniques: Expressing, Activation Assay, De-Phosphorylation Assay, Ligation

Journal: Developmental Biology

Article Title: Rab3Gap1 mediates exocytosis of Claudin-1 and tight junction formation during epidermal barrier acquisition

doi: 10.1016/j.ydbio.2013.04.034

Figure Lengend Snippet: Ppp2r2a knockdown cells have a defective paracellular barrier and altered cell surface expression of tight junction components. (A) Haemotoxilin and Eosin stain of scrambled control and Ppp2r2a kd cultures 3,5,7 and 10 days post raising to the air–liquid interface. Lower 2 rows are the corresponding whole mount haematoxylin staining, showing delay in outside-in barrier acquisition in the Ppp2r2a kd organotypic cultures. (B) 4 kDa FITC-dextran penetration in post-confluent cells. Error bars are s.d. for triplicate results, and data is representative of n =6 experiments. (C) Confocal microscopy of Immunofluoresence staining of pcJun, Total cJun, Claudin-1, Zo-1 and Occludin in organotypic culture. (D) Immunofluoresence of Claudin-1, Zo-1 and Occludin in scrambled control and Ppp2r2a kd cells in post-confluent culture. (E) Western blot of Ppp2r2a, Occludin and Claudin-1, and other Tight junction components differentially expressed in Ppp2r2a kd cells, confirming Ppp2r2a knockdown and the up-regulation of Claudin-1 and Jam-A. Bar 100 µm (A), 50 µm (C), and 10 µm(D).

Article Snippet: Primary antibodies and concentrations were as follows: rabbit anti PR55a (Ppp2r2a) (Santa Cruz Biotechnologies, Santa Cruz, US) 1/500; rabbit anti cJun (Cell Signalling Technologies, Danvers, USA) 1/100; rabbit anti pSer63 cJun (Cell Signalling Technologies, Danvers, USA) 1/100; rabbit anti Claudin-1 (Invitrogen, Paisley, UK) 1/500; mouse anti Occludin (Invitrogen, Paisley, UK ) 1/500; Rabbit anti Zonula Occludins-1 (Santa Cruz Biotechnologies, Santa Cruz, US) 1/500; Mouse anti Rab3 (BD biosciences, Oxford, UK) 1/100.

Techniques: Knockdown, Expressing, Staining, Control, Confocal Microscopy, Western Blot

Journal: Developmental Biology

Article Title: Rab3Gap1 mediates exocytosis of Claudin-1 and tight junction formation during epidermal barrier acquisition

doi: 10.1016/j.ydbio.2013.04.034

Figure Lengend Snippet: Jun Kinase inhibition restores cell surface expression of tight junction components in Ppp2r2a siRNA expressing cells. (A) 4 kDa FITC dextran penetration experiment at 5 days post confluence comparing scrambled controls, Ppp2r2a kd cells alone or treated with the JNK inhibitor SP600125. *, p -value 0.001, Two-tailed t -test. Error bars are s.d. (B) pcJun and Claudin-1 immunofluoresence in Scrambled controls, siRNA expressing alone or treated with SP600125, showing restoration of normal levels of pcJun and normal localisation of Claudin-1. This correlated with a reduction in hyperkeratosis in the SP600125 treated organotypics. (C) Claudin-, Zo-1 and Occludin immunofluorescence in Ppp2r2a siRNA kd cells alone or treated with SP600125. (D) Western blot analysis of Occludin, Claudin and Zo-1 in the same cells. Gapdh is the loading control. Numbers indicate the fold increase compared to scrambled controls. E. Proximity ligation analysis of Claudin-1 and Occludin and Zo-1 and Occludin interaction in Ppp2r2a kd cells and Ppp2r2a kd cells treated with the Jun Kinase inhibitor SP600125, (* p <0.05, ** p <0.001, n =3). Bars 50 µm (B) and 10 µm (C, E).

Article Snippet: Primary antibodies and concentrations were as follows: rabbit anti PR55a (Ppp2r2a) (Santa Cruz Biotechnologies, Santa Cruz, US) 1/500; rabbit anti cJun (Cell Signalling Technologies, Danvers, USA) 1/100; rabbit anti pSer63 cJun (Cell Signalling Technologies, Danvers, USA) 1/100; rabbit anti Claudin-1 (Invitrogen, Paisley, UK) 1/500; mouse anti Occludin (Invitrogen, Paisley, UK ) 1/500; Rabbit anti Zonula Occludins-1 (Santa Cruz Biotechnologies, Santa Cruz, US) 1/500; Mouse anti Rab3 (BD biosciences, Oxford, UK) 1/100.

Techniques: Inhibition, Expressing, Two Tailed Test, Immunofluorescence, Western Blot, Control, Ligation

Journal: Developmental Biology

Article Title: Rab3Gap1 mediates exocytosis of Claudin-1 and tight junction formation during epidermal barrier acquisition

doi: 10.1016/j.ydbio.2013.04.034

Figure Lengend Snippet: Genes involved in exocytosis and the regulation of ubiquitin-dependent proteolysis are altered in Ppp2r2a siRNA expressing cells and restored by Jun Kinase inhibition. (A) Clusters of differentially expressed genes were identified in the Ppp2r2a kd cells, both non-rescued and rescued by SP600125 treatment. The number in the corner is the number of differentially expressed genes in that cluster (% of all differentially expressed genes). (B) Graphical representation of over-represented gene ontology groups in differentially expressed and SP600125 rescued genes, groups with an asterisk were investigated further. The blue line denotes the p <0.05 significance value. (C) Table of the differentially expressed genes in each of the indicated gene ontology groups. Fold reduction in the Ppp2r2a kd cells is shown in brackets. (D and E) STRING networks of interacting down-regulated and SP600125 rescued genes involved in the proteasome complex (D) and Exocytosis (E).

Article Snippet: Primary antibodies and concentrations were as follows: rabbit anti PR55a (Ppp2r2a) (Santa Cruz Biotechnologies, Santa Cruz, US) 1/500; rabbit anti cJun (Cell Signalling Technologies, Danvers, USA) 1/100; rabbit anti pSer63 cJun (Cell Signalling Technologies, Danvers, USA) 1/100; rabbit anti Claudin-1 (Invitrogen, Paisley, UK) 1/500; mouse anti Occludin (Invitrogen, Paisley, UK ) 1/500; Rabbit anti Zonula Occludins-1 (Santa Cruz Biotechnologies, Santa Cruz, US) 1/500; Mouse anti Rab3 (BD biosciences, Oxford, UK) 1/100.

Techniques: Ubiquitin Proteomics, Expressing, Inhibition

Journal: Developmental Biology

Article Title: Rab3Gap1 mediates exocytosis of Claudin-1 and tight junction formation during epidermal barrier acquisition

doi: 10.1016/j.ydbio.2013.04.034

Figure Lengend Snippet: Jun Kinase inhibition restores Rab3Gap1 expression and Claudin-1 cell surface expression. Cell surface Rab3Gap1 correlates with barrier acquisition during development. A. Western blot analysis of Rab3Gap1 and Rab3 and Exoc3 in response to SP600125 treatment in Ppp2r2a kd cells. Numbers denote fold change in intensity compared to Scrambled controls after normalisation by levels of Gapdh, the loading control. (B) Western blot of Rab3Gap1, Rab3 and Exoc3 in REKs treated with SP600125 over an 8 h period. Note the reduction in phosphorylated cJun (pcJun) from 30 min correlates with the induction of Rab3Gap1 C. Immunofluorescence of Rab3Gap1 and Co-immunofluorescence of Claudin-1 and Rab3 in confluent scrambled, Ppp2r2a kd cells and Ppp2r2a kd cells treated with SP600125. D. Immunofluorescence of pcJun, pSerAkt, Ppp2r2a, Claudin-1 and Rab3Gap1 in E15.5 (Pre-barrier) and E17.5 (Post barrier) mouse epidermis, showing cell surface localisation of both Rab3Gap1 and Claudin-1 post barrier acquisition. Dotted line indicates the dermal epidermal boundary. Bar 10 µm (C) and 50 µm (D).

Article Snippet: Primary antibodies and concentrations were as follows: rabbit anti PR55a (Ppp2r2a) (Santa Cruz Biotechnologies, Santa Cruz, US) 1/500; rabbit anti cJun (Cell Signalling Technologies, Danvers, USA) 1/100; rabbit anti pSer63 cJun (Cell Signalling Technologies, Danvers, USA) 1/100; rabbit anti Claudin-1 (Invitrogen, Paisley, UK) 1/500; mouse anti Occludin (Invitrogen, Paisley, UK ) 1/500; Rabbit anti Zonula Occludins-1 (Santa Cruz Biotechnologies, Santa Cruz, US) 1/500; Mouse anti Rab3 (BD biosciences, Oxford, UK) 1/100.

Techniques: Inhibition, Expressing, Western Blot, Control, Immunofluorescence

Journal: Developmental Biology

Article Title: Rab3Gap1 mediates exocytosis of Claudin-1 and tight junction formation during epidermal barrier acquisition

doi: 10.1016/j.ydbio.2013.04.034

Figure Lengend Snippet: Knockdown of Rab3Gap1 prevents cell surface localisation of Claudin-1 and Occludin. Rab3Gap1 rescues Claudin-1 localisation and expression in Ppp2r2a kd cells. ( A) Immunofluoresence of Rab3Gap1 in Scrambled controls and in two shRNA knockdowns of Rab3Gap1 (siRNA1 and siRNA4). (B) Western blot of Rab3Gap1 kd cells of Rab3Gap1, Claudin-1, Rab3a, Occludin, Zo-1, Exoc3 and Psmd4. Gapdh is the loading control. (C) Graph showing the mean percentage intensity in both kd lines (siRNA) compared to Scrambled controls (Scr) after normalisation by Gapdh. Error bars SD, *, p <0.05 **, p <0.005 (2-sided Students T-Test). (D) co-immunofluorescence of Claudin-1 and Rab3, showing peri-nuclear co-localisation in post-confluent Rab3Gap1 kd cells. E. Immunofluorescent analysis of Rab3Gap1 in confluent scrambled and Rab3Gap1 kd confluent keratinocytes. F. Expression of Claudin-1 in confluent scrambled controls, Ppp2r2a kd cells and kd cells transiently transfected with Rab3Gap1 (representative of 2 separate transfections). G. Western blot of Scrambled, Ppp2r2a kd cells and kd cells transfected with a Myc-tagged Rab3Gap1 (arrowhead). Numbers denote fold change in intensity of Claudin-1, Exoc3 and Rab3Gap1 compared to Scrambled controls after normalisation by levels of Gapdh, the loading control Bar 10 µm (A, D, E, F), 50 µm (E (low mag view)).

Article Snippet: Primary antibodies and concentrations were as follows: rabbit anti PR55a (Ppp2r2a) (Santa Cruz Biotechnologies, Santa Cruz, US) 1/500; rabbit anti cJun (Cell Signalling Technologies, Danvers, USA) 1/100; rabbit anti pSer63 cJun (Cell Signalling Technologies, Danvers, USA) 1/100; rabbit anti Claudin-1 (Invitrogen, Paisley, UK) 1/500; mouse anti Occludin (Invitrogen, Paisley, UK ) 1/500; Rabbit anti Zonula Occludins-1 (Santa Cruz Biotechnologies, Santa Cruz, US) 1/500; Mouse anti Rab3 (BD biosciences, Oxford, UK) 1/100.

Techniques: Knockdown, Expressing, shRNA, Western Blot, Control, Immunofluorescence, Transfection

Journal: Brain Pathology

Article Title: Axonopathy Is Associated with Complex Axonal Transport Defects in a Model of Multiple Sclerosis

doi: 10.1111/j.1750-3639.2011.00541.x

Figure Lengend Snippet: Antibodies used for immunohistochemistry and immunofluorescence. Abbreviations: IHC = immunohistochemistry; IF = immunofluorescence; PBS = phosphate‐buffered saline; NA = not applicable; ND = not done.

Article Snippet: 5. , Protein phosphatase 2 subunit A (Ppp2r2a) , Cell Signaling Technology , 2041 , Rabbit , 1:50 , ND , Boiled in citrate buffer in the microwave.

Techniques: Immunohistochemistry, Immunofluorescence, Saline, Ubiquitin Proteomics

Journal: Brain Pathology

Article Title: Axonopathy Is Associated with Complex Axonal Transport Defects in a Model of Multiple Sclerosis

doi: 10.1111/j.1750-3639.2011.00541.x

Figure Lengend Snippet: List of axonal transport‐related differentially expressed genes.

Article Snippet: 5. , Protein phosphatase 2 subunit A (Ppp2r2a) , Cell Signaling Technology , 2041 , Rabbit , 1:50 , ND , Boiled in citrate buffer in the microwave.

Techniques: Infection, Targeted Proteomics, Ubiquitin Proteomics