Journal:

Article Title: Amino Acid Substitutions at Ambler Position Gly238 in the SHV-1 ?-Lactamase: Exploring Sequence Requirements for Resistance to Penicillins and Cephalosporins

doi: 10.1128/AAC.46.12.3971-3977.2002

Figure Lengend Snippet: Western blot and slot blot of SHV-1 β-lactamase and mutants at the 238 position. The Western blot and slot blot were probed with 1 μg of anti-SHV-1 antibody/ml and horseradish peroxidase-conjugated protein G (7). Single-letter designations for the Gly238 mutant β-lactamases are listed above each slot. SK− represents the strain E. coli DH10B with the vector pBCSK(−) without the SHV β-lactamase. (Inset) Western blot of SHV-1 and Gly238Ala variant of the SHV β-lactamase. Equal amounts of purified β-lactamase were loaded in each lane. Lane 1, SHV-1; lane 2, Gly238Ala. As evaluated by densitometry, SHV-1 and Gly238Ala are nearly equivalent.

Article Snippet: Each of the strains possessing the 19 variants and the wild-type β-lactamase were assayed for in vivo steady-state expression levels by probing them with 1 μg/ml of purified anti-SHV antibody and horseradish peroxidase-conjugated protein G (Bio-Rad) as previously reported ( 7 ).

Techniques: Western Blot, Dot Blot, Mutagenesis, Plasmid Preparation, Variant Assay, Purification

Journal: Frontiers in Molecular Neuroscience

Article Title: Cloning and Phylogenetic Analysis of NMDA Receptor Subunits NR1, NR2A and NR2B in Xenopus laevis Tadpoles

doi: 10.3389/neuro.02.004.2009

Figure Lengend Snippet: Detection of NMDAR subunits and splice variants in X. laevis tadpole brain . (A) Immunoblots for NR1, NR2A and NR2B were done on whole brain lysates of stage 47/48 tadpoles (Xen) with antibodies generated against homologous rat proteins. Immunoreactive bands of about the same size as in rat whole brain lysate (Rat) were found for all three subunits. (B) Western blots of whole cell lysates of HEK293 cells transfected with X. laevis the NR1-4a/b splice variant or NR2A cDNA, and their mock-transfected controls. (C) Cartoon of NR1 splice variants and the alternatively spliced exons (N1, C1, C2) and C-terminal ends (C2′) that they contain. (D) Cross-reactive bands for exons N1 and C1, as well as alternative C-terminal end C2′ in X. laevis were detected with antibodies to rat homologs by Western blot on whole brain extracts from stage 47/48 tadpoles and rat. No cross-reactive band was detected for C2. (E,F) C-terminal ends of NR1 mRNAs were amplified by RT-PCR and PCR with primer pairs 5′ to the spliced region and in the 3′UTR. (E) Twenty-six out of 29 nucleotide sequences contained only C2′ (Xen_NR1_C2′). They aligned perfectly to the published X. laevis NR1 sequence (Xen_NR1_X94156). (F) Two out of 29 nucleotide sequences contained C1 and C2′ (Xen_NR1_C1_C2′), indicating that NR1-3a/b also exists in X. laevis . The alignment to rat NR1-3a (Rat_NR1-3a) shows a very high degree of sequence conservation. Identical residues are marked with (*).

Article Snippet: An equal amount of protein per lane was separated by 8% SDS-PAGE and transferred onto nitrocellulose membrane that were probed with primary antibodies at 1:100–500: α-NR1, α-NR2B (both BD PharMingen), α-NR2A (crude rabbit serum JH 1817, gift from the Huganir Lab), α-NR1-C1 (crude rabbit serum JH 2079, gift from the Huganir Lab), α-NR1-N1, α-NR1-C2 and α-NR1-C2’ (all three Novus Biologicals) and incubated with the HRP-conjugated α-goat or α-mouse secondary antibody (Bio-Rad, 1:3000).

Techniques: Western Blot, Generated, Transfection, Variant Assay, Amplification, Reverse Transcription Polymerase Chain Reaction, Sequencing