Journal:

Article Title: Flagellar Radial Spoke Protein 2 Is a Calmodulin Binding Protein Required for Motility in Chlamydomonas reinhardtii

doi: 10.1128/EC.3.1.72-81.2004

Figure Lengend Snippet: (A) Strategy for cloning RSP2 starting with peptide sequences from band-purified RSP2 (see Materials and Methods). PCR, using the degenerate oligonucleotide primers (arrows) derived from two peptides, resulted in a probe for screening cDNA and genomic libraries. Additional RT-PCR was carried out to recover entire cDNA. The coding sequence (gray boxes), untranslated regions (open boxes), and introns (black boxes) were deduced by comparing complete genomic, cDNA, and peptide sequences. (B) Northern blot analysis, with the RSP2 RT-PCR probe, revealed a 2.7-kb message that became greatly amplified in cells 45 min following deflagellation (R, RNA from flagellar regenerating cells; NR, RNA from control, nonregenerating cells). (C) Predicted amino acid sequences of RSP2 (accession no. AY373262). Sequences obtained by peptide microsequencing are underlined. Arrows indicate the peptide sequences used for designing degenerate oligonucleotide primers for successful RT-PCR, and boldface italic indicates amphipathic helical domains predicted to bind calmodulin (Fig. ​(Fig.55 and ​and6).6). (D) Comparison of wild-type and pf24 RSP2 genomic sequences, revealing that the pf24 mutation results from a single base pair replacement and conversion of the initial ATG to ATA. WT, wild type.

Article Snippet: For calmodulin affinity purification of the recombinant RSP2 (see below), calmodulin-Sepharose was used according to the instructions of the manufacturer (Amersham Pharmacia, Piscataway, N.J.).

Techniques: Clone Assay, Purification, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Sequencing, Northern Blot, Amplification, Genomic Sequencing, Mutagenesis

Journal:

Article Title: Flagellar Radial Spoke Protein 2 Is a Calmodulin Binding Protein Required for Motility in Chlamydomonas reinhardtii

doi: 10.1128/EC.3.1.72-81.2004

Figure Lengend Snippet: Predicted tertiary structure of RSP2. (A) Sequence analysis with the SMART program predicted four coiled-coil domains, a GAF domain, a hexokinase-like domain, and two basic amphipathic helical structures predicted to bind calmodulin. (B) Sequence comparison of the homologous regions in hexokinase and the C-terminal half of the RSP2 GAF domain predicted by the Blast-PSI program; similar residues are shown by gray boxes, and identical residues are shown by black boxes. (C) The two basic amphipathic helices were identified based on the Eisenberg moment and plotted as a helical wheel with the Protean program. Basic amino acid residues are designated by gray circles. (D) The basic amphipathic helices contain the conserved hydrophobic residues (boldface) and multiple basic residues characteristic of type A (residues 579 to 592) and type B (residues 546 to 559) 1-8-14 calcium-dependent calmodulin binding motifs (45).

Article Snippet: For calmodulin affinity purification of the recombinant RSP2 (see below), calmodulin-Sepharose was used according to the instructions of the manufacturer (Amersham Pharmacia, Piscataway, N.J.).

Techniques: Sequencing, Binding Assay

Journal:

Article Title: Flagellar Radial Spoke Protein 2 Is a Calmodulin Binding Protein Required for Motility in Chlamydomonas reinhardtii

doi: 10.1128/EC.3.1.72-81.2004

Figure Lengend Snippet: Recombinant RSP2 binds calmodulin in a Ca2+-dependent manner. (A) Extracts from bacteria transformed with the expression constructs for either native or six-His-tagged RSP2 were analyzed by Western blotting with monoclonal (left panel) and polyclonal (right panel) anti-RSP2 antibodies. Axonemes from wild-type and pf14 cells (lane 2, right panel) and the bacterial extracts without the fusion proteins (lane 4, right panel) served as controls. Compared to the polyclonal antibody (right panel), the monoclonal antibody (left panel) recognized the recombinant protein with significantly lower affinity. WT, wild type. (B) Calmodulin affinity chromatography was performed on extracts from the bacteria expressing native RSP2 in the presence (middle panel, Coomassie blue protein stain; right panel, parallel Western blot with RSP2 antibody) and absence (left panel) of calcium, and then the proteins were eluted from the calmodulin matrix with 2 mM EGTA (“+” symbol, all panels). In the presence of EGTA, no binding of RSP2 was detected (arrow, left panel). In contrast, when Ca2+ was present, RSP2 bound to the matrix (compare “pre” and “post” columns, middle and right panels), and in comparison, no bacterial proteins were visibly reduced following extraction with the calmodulin-Sepharose matrix. A fraction of the bound RSP2 can be eluted with EGTA (compare “−” and “+” lanes, middle and right panels), including degradation products of the expressed RSP2 revealed by Western blotting (right panel). However, a large fraction of the bound RSP2 remained associated with the calmodulin matrix following EGTA elution (“beads,” middle and right panels), indicating strong affinity between a fraction of RSP2 and calmodulin. Numbers at left of the middle panel are molecular masses in kilodaltons.

Article Snippet: For calmodulin affinity purification of the recombinant RSP2 (see below), calmodulin-Sepharose was used according to the instructions of the manufacturer (Amersham Pharmacia, Piscataway, N.J.).

Techniques: Recombinant, Transformation Assay, Expressing, Construct, Western Blot, Affinity Chromatography, Staining, Binding Assay

Journal: Frontiers in Oncology

Article Title: Suppression of EGFR/PKC-δ/NF-κB Signaling Associated With Imipramine-Inhibited Progression of Non-Small Cell Lung Cancer

doi: 10.3389/fonc.2021.735183

Figure Lengend Snippet: The inhibition of EGFR/PKC-δ/NF-κB proteins phosphorylation and induction of apoptosis-related proteins by imipramine in CL1-5-F4/ NF-κB-luc2 bearing tumor. (A–C) The protein expression from IHC of EGFR (Try 1068), PKC-δ (Thr507), NF-κB (Ser536), cleaved caspase-3, -8, -9, MMP-9, XIAP, MCL-1 and their quantification bar chart are presented. (D, E) The tumor ex vivo Western blotting from each mice of cleaved caspase-3, -8, -9 and PARP-1 is presented. ( ** p < 0.01 vs . vehicle; scale bar =100 μm).

Article Snippet: Primary antibodies against Matrix metalloproteinase-9 (MMP-9) (AB19016, Millipore), vascular endothelial growth factor (VEGF) (ab1316, Abcam, Cambridge, UK), EGFR (Try 1068) (#2234, Cell signaling, Danvers, MA, USA), EGFR (E-AB-63555, Elabscience, Houston, TX, USA), PKC-δ (Thr507) (E-AB-20968, Elabscience), PKC-δ (E-AB-14675, Elabscience), NF-κB p65 (Ser536) (E-AB-70335, Elabscience), NF-κB p65 (E-AB-22066, Elabscience), cell leukemia-1 (MCL-1) (BV-438, BioVision), cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (cFLIP) (D16A8, Cell signaling), X-linked inhibitor of apoptosis protein (XIAP) (PA5-29253, Thermo Fisher Scientific), Fas (E-AB-40063, Elabscience), Fas ligand (FasL) (E-AB-31410, Elabscience), cleaved caspase-3 (E-AB-30004, Elabscience), cleaved caspase-8 (E-AB-22107, Elabscience), cleaved caspase-9 (#9505, Cell Signaling Technology), PARP-1 (#9532, Cell Signaling Technology) and β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, Texas, Waltam, MA, USA) for Western blotting were purchased from different companies as listed.

Techniques: Inhibition, Phospho-proteomics, Expressing, Ex Vivo, Western Blot