File S1 - Direct Interaction between AR and PAK6 in Androgen-Stimulated PAK6 Activation

Supporting Figures S1-S6.
Figure S1. Identification of Serine-308 as a site of phosphorylation. The amino acid sequence is provided above the spectrum, and the masses above and below the sequence correspond to the theoretical b- and y-type product ions, respectively. The masses provided are the singly-protonated, monoisotopic product ion masses. The observed singly-protonated product ions are underlined. For simplicity, all doubly protonated ions are not labeled in the spectra as they exist at 50% abundance or less. Asterisks indicate ions that result from neutral loss of H3PO4.
Figure S2. Identification of Threonine-326 as a site of phosphorylation. The amino acid sequence is provided above the spectrum, and the masses above and below the sequence correspond to the theoretical b- and y-type product ions, respectively. The masses provided are the singly-protonated, monoisotopic product ion masses. The observed singly-protonated product ions are underlined. Asterisks indicate ions that result from neutral loss of H3PO4 from fragment ions. Additionally, the presence of the second site of phosphorylation is determined due to accurate mass of the peptide, however, the first fragment ion representing this site is located at y7, thus the phosphorylation could exist in any of the sites shown in brackets.
Figure S3. Identification of Serine-346 as a site of phosphorylation. The amino acid sequence is provided above the spectrum, and the masses above and below the sequence correspond to the theoretical b- and y-type product ions, respectively. The masses provided are the singly-protonated, monoisotopic product ion masses. The observed singly-protonated product ions are underlined. The site of phosphorylation cannot be definetively identified, due to lack of specific ions related to either site of phosphorylation, however we hypothesize that the phosphorylation is on the first of the serine residues due to lack of tryptic cleavage at the preceding site. Asterisks indicate ions that result from neutral loss of H3PO4.
Figure S4. Identification of Thrionine-354 and Serine-360 as sites of phosphorylation. The amino acid sequence is provided above the spectrum, and the masses above and below the sequence correspond to the theoretical b- and y-type product ions, respectively. The masses provided are the singly-protonated, monoisotopic product ion masses. The observed singly-protonated product ions are underlined. Asterisks indicate ions that result from neutral loss of H3PO4 from fragment ions. This doubly phosphorylated peptide loses two phosphoric acid groups from the parent ion which are marked with *.
Figure S5. Identification of Serine-360 as a site of phosphorylation. The amino acid sequence is provided above the spectrum, and the masses above and below the sequence correspond to the theoretical b- and y-type product ions, respectively. The masses provided are the singly-protonated, monoisotopic product ion masses. The observed singly-protonated product ions are underlined. Asterisks indicate ions that result from neutral loss of H3PO4 from fragment ions.
Figure S6. Identification of Serine560 as a site of phosphorylation. The amino acid sequence is provided above the spectrum, and the masses above and below the sequence correspond to the theoretical b- and y-type product ions, respectively. The masses provided are the singly-protonated, monoisotopic product ion masses. The observed singly-protonated product ions are underlined. Asterisks indicate ions that result from neutral loss of H3PO4.
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补充图S1至S6。
图S1：丝氨酸-308（Serine-308）磷酸化位点的鉴定。谱图上方标注了氨基酸序列，序列上下两侧的质量数分别对应理论b型和y型产物离子。所给出的质量数为单质子化单同位素产物离子质量。观测到的单质子化产物离子已以下划线标注。为简化展示，丰度≤50%的双质子化离子未在谱图中标注。星号代表因H₃PO₄中性丢失产生的离子。
图S2：苏氨酸-326（Threonine-326）磷酸化位点的鉴定。谱图上方标注了氨基酸序列，序列上下两侧的质量数分别对应理论b型和y型产物离子。所给出的质量数为单质子化单同位素产物离子质量。观测到的单质子化产物离子已以下划线标注。星号代表由片段离子发生H₃PO₄中性丢失产生的离子。此外，根据肽段的精确质量可确认存在第二个磷酸化位点，但代表该位点的首个片段离子为y₇，因此磷酸化位点可能位于方括号标注的任意残基中。
图S3：丝氨酸-346磷酸化位点的鉴定。谱图上方标注了氨基酸序列，序列上下两侧的质量数分别对应理论b型和y型产物离子。所给出的质量数为单质子化单同位素产物离子质量。观测到的单质子化产物离子已以下划线标注。由于缺乏与任一候选磷酸化位点相关的特征离子，无法明确鉴定该磷酸化位点；但鉴于前一位点未发生胰蛋白酶裂解，我们推测磷酸化发生在首个丝氨酸残基上。星号代表因H₃PO₄中性丢失产生的离子。
图S4：苏氨酸-354与丝氨酸-360磷酸化位点的鉴定。谱图上方标注了氨基酸序列，序列上下两侧的质量数分别对应理论b型和y型产物离子。所给出的质量数为单质子化单同位素产物离子质量。观测到的单质子化产物离子已以下划线标注。星号代表由片段离子发生H₃PO₄中性丢失产生的离子。该双磷酸化肽段会从母离子上丢失两个磷酸基团，分别以*标注。
图S5：丝氨酸-360磷酸化位点的鉴定。谱图上方标注了氨基酸序列，序列上下两侧的质量数分别对应理论b型和y型产物离子。所给出的质量数为单质子化单同位素产物离子质量。观测到的单质子化产物离子已以下划线标注。星号代表由片段离子发生H₃PO₄中性丢失产生的离子。
图S6：丝氨酸-560磷酸化位点的鉴定。谱图上方标注了氨基酸序列，序列上下两侧的质量数分别对应理论b型和y型产物离子。所给出的质量数为单质子化单同位素产物离子质量。观测到的单质子化产物离子已以下划线标注。星号代表因H₃PO₄中性丢失产生的离子。
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