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Cytoskeletal rearrangements in human red blood cells induced by snake venoms: light microscopy of shapes and NMR studies of membrane function
Tsz Wai Yau*†, Rhiannon P. Kuchel‡, Jennifer M. S. Koh*, David Szekely§, Peter J. Mirtschin¶ and Philip W. Kuchel1*‖
*School of Molecular Bioscience, University of Sydney, Sydney, NSW 2006, Australia, †Australian Nuclear Science and Technology Organisation, Lucas Heights, Sydney, NSW 2232, Australia, ‡Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia, §Mark Cowley Lidwill Program in Cardiac Electrophysiology, The Victor Chang Cardiac Research Institute, Sydney, NSW 2010, Australia, ¶School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5001, Australia, and ‖Singapore Bioimaging Consortium, BioMedical Sciences Institutes, Biopolis, Singapore 138667, Singapore
1To whom correspondence should be addressed (email firstname.lastname@example.org).
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Figure S1 SDS/PAGE gel analysis and immunoblot analysis of RBC membrane proteins before and after snake venom treatment
(A) SDS/PAGE. Lane 1, membrane proteins from control RBCs without exposure to snake venom. Lane 2, membrane proteins from RBCs that had been treated with 0.1 mg/ml of P. guttatus venom. There was no significant difference in the concentration of protein components (based on protein band intensity and area). Membrane proteins were extracted by the method described in Section 2.2 of the main text. The SDS/PAGE gel was run at 150 V for 80 min, then stained for 1 h in 0.1% Coomassie Brilliant Blue R250 with 10% (v/v) methanol and 10% (v/v) acetic acid, and de-stained overnight in 10% (v/v) methanol and 7% (v/v) acetic acid before being photographed. (B) Immunoblot showing various levels of α-spectrin, β-spectrin, Band 3 and actin in two suspensions of RBCs; control RBCs and snake venom treated RBCs. The protein level of venom-treated RBC membrane samples of different incubation times, 30 min and 60 min, were compared with control RBCs using the immunoblot experiment. The results showed that there was no significant difference in protein concentration upon venom treatment, as well as confirming the identity of the proteins. It was also shown that there were no differences in protein concentration even with extended incubation times. The samples (10 μg of protein) were loaded onto each well of a 4–12% (w/v) SDS/PAGE gel. SeeBlue® Plus2 (Invitrogen) was used as the protein size standard. The gel was run for 3 h at 150 V on ice, and proteins from the gel were transferred to a nitrocellulose membrane, and then it was run at 30 V overnight in a transfer buffer (NuPAGE, Invitrogen) kept at 4°C. The membrane was blocked with 5% (w/v) non-fat dried skimmed milk powder in TBST (Tris-buffered saline plus Tween) for 1 h at room temperature. From hereafter, the blotting incubations were all performed at room temperature. The primary antibodies of anti-α-spectrin, anti-β-spectrin, anti-Band 3, or anti-actin were diluted 1:4000, 1:500, 1:1500 and 1:500 respectively before being incubated with the membrane while rocking for 1 h. The membrane was washed 3 times for 10 min in TBST while rocking. The secondary antibody, of anti-mouse linked to peroxidase or anti-goat IgG linked to peroxidase, was diluted 1:20000 and was then incubated with the membrane while rocking for 1 h. The membrane was washed 3 times for 10 min in TBST while rocking. Immobilon Western chemiluminescent horseradish peroxidase substrates (Millipore) were prepared, and the nitrocellulose membrane was incubated for 5 min at room temperature. Photographic films were exposed for 15–120 s before developing.
Figure S2 DIC micrographs of two separate RBC suspensions incubated with various snake venoms for 2 h, at 25°C
(A) RBC suspension treated with P. guttatus venom (0.1 mg/ml). (B) RBC suspension treated with a mixture of venoms from P. guttatus (0.1 mg/ml) and O. scutellatus (0.1 mg/ml). (C) Control RBC suspension without any treatment. The results showed that O. scutellatus venom reduced the haemolytic activity of P. guttatus venom.
Figure S3 DIC micrographs of two separate RBC suspensions incubated with snake venom or pre-heated snake venom for 2 h at 25°C
(A) RBC suspension treated with P. guttatus venom (1 mg/ml) for 2 h. (B) RBC suspension treated with P. guttatus venom (1 mg/ml) which was heated to 90°C for 15 min prior to addition to the RBC suspension; the micrographs showed that no snake venom-induced morphologies appeared.
Figure S4 DIC micrographs of two separate RBC suspensions incubated with higher molecular mass or lower molecular mass snake venom components for 2 h at 25°C
(A) Micrograph taken at the end of the incubation of an RBC suspension with higher molecular mass proteins of P. guttatus venom. (B) Micrograph taken at the end of the incubation of an RBC suspension treated with lower molecular mass proteins of P. guttatus venom. The higher and lower molecular mass proteins were separated using a Sephadex G75 column by the method described in Section 2.8 of the main text. Results from this experiment showed that the shape-changing activity existed amongst the lower molecular mass components.
Received 8 January 2011/7 July 2011; accepted 21 September 2011
Published as Cell Biology International Immediate Publication 21 September 2011, doi:10.1042/CBI20110012
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